Maternal and fetal human leukocyte antigen class Ia and II alleles in severe preeclampsia and eclampsia.

A line of investigations indicate that genes in the human leukocyte antigen (HLA) complex are involved in a successful acceptance of the semiallogeneic fetus during pregnancy. In this study, associations between specific HLA class Ia (HLA-A and -B) and class II (HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1) alleles and the risk of developing severe preeclampsia/eclampsia were investigated in a detailed and large-scale study. In total, 259 women diagnosed with severe preeclampsia or eclampsia and 260 matched control women with no preeclampsia, together with their neonates, were included in the study. HLA genotyping for mothers and neonates was performed using next-generation sequencing. The HLA-DPB1*04:01:01G allele was significantly more frequent (Pc=0.044) among women diagnosed with severe preeclampsia/eclampsia compared with controls, and the DQA1*01:02:01G allele frequency was significantly lower (Pc=0.042) among newborns born by women with severe preeclampsia/eclampsia compared with controls. In mothers with severe preeclampsia/eclampsia, homozygosity was significantly more common compared with controls at the HLA-DPB1 locus (Pc=0.0028). Although the current large study shows some positive results, more studies, also with a functional focus, are needed to further clarify a possible role of the classical HLA genes in preeclampsia.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1.

The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified. Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

Genomic differences between Fibrobacter succinogenes S85 and Fibrobacter intestinalis DR7, identified by suppression subtractive hybridization.

Fibrobacter is a highly cellulolytic genus commonly found in the rumen of ruminant animals and cecum of monogastric animals. In this study, suppression subtractive hybridization was used to identify the genes present in Fibrobacter succinogenes S85 but absent from F. intestinalis DR7. A total of 1,082 subtractive clones were picked, plasmids were purified, and inserts were sequenced, and the clones lacking homology to F. intestinalis were confirmed by Southern hybridization. By comparison of the sequences of the clones to one another and to those of the F. succinogenes genome, 802 sequences or 955 putative genes, comprising approximately 409 kb of F. succinogenes genomic DNA, were identified that lack similarity to those of F. intestinalis chromosomal DNA. The functional groups of genes, including those involved in cell envelope structure and function, energy metabolism, and transport and binding, had the largest number of genes specific to F. succinogenes. Low-stringency Southern hybridization showed that at least 37 glycoside hydrolases are shared by both species. A cluster of genes responsible for heme, porphyrin, and cobalamin biosynthesis in F. succinogenes S85 was either missing from or not functional in F. intestinalis DR7, which explains the requirement of vitamin B12 for the growth of the F. intestinalis species. Two gene clusters encoding NADH-ubiquinone oxidoreductase subunits probably shared by Fibrobacter genera appear to have an important role in energy metabolism.

Draft Genome Sequence of Cyanobacterium sp. Strain HL-69, Isolated from a Benthic Microbial Mat from a Magnesium Sulfate-Dominated Hypersaline Lake.

The complete genome sequence of Cyanobacterium sp. strain HL-69 consists of 3,155,247 bp and contains 2,897 predicted genes comprising a chromosome and two plasmids. The genome is consistent with a halophilic nondiazotrophic phototrophic lifestyle, and this organism is able to synthesize most B vitamins and produces several secondary metabolites.

Geochemical and Microbial Community Attributes in Relation to Hyporheic Zone Geological Facies.

The hyporheic zone (HZ) is the active ecotone between the surface stream and groundwater, where exchanges of nutrients and organic carbon have been shown to stimulate microbial activity and transformations of carbon and nitrogen. To examine the relationship between sediment texture, biogeochemistry, and biological activity in the Columbia River HZ, the grain size distributions for sediment samples were characterized to define geological facies, and the relationships among physical properties of the facies, physicochemical attributes of the local environment, and the structure and activity of associated microbial communities were examined. Mud and sand content and the presence of microbial heterotrophic and nitrifying communities partially explained the variability in many biogeochemical attributes such as C:N ratio and %TOC. Microbial community analysis revealed a high relative abundance of putative ammonia-oxidizing Thaumarchaeota and nitrite-oxidizing Nitrospirae. Network analysis showed negative relationships between sets of co-varying organisms and sand and mud contents, and positive relationships with total organic carbon. Our results indicate grain size distribution is a good predictor of biogeochemical properties, and that subsets of the overall microbial community respond to different sediment texture. Relationships between facies and hydrobiogeochemical properties enable facies-based conditional simulation/mapping of these properties to inform multiscale modeling of hyporheic exchange and biogeochemical processes.

Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae.

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Uptake and decay of volatile organic compounds at environmental concentrations: application of a four-compartment model to a chamber study of five human subjects.

Five subjects were exposed to nine volatile organic compounds (VOCs) at concentrations that can be encountered in everyday life. Breath samples were collected during a 10-h uptake phase and a 24-h decay phase. It was possible to determine four distinct slopes in the decay curve for each chemical. The distribution in the body and residence times in different tissues were calculated using a linear four-compartment mass-balance model. The model was used to predict breath concentrations for two subjects in a second chamber experiment including the same nine VOCs, representing three chemical classes (aromatic, aliphatic, and chlorinated compounds). Predicted values were generally within 25% of those observed, suggesting that the model parameters calculated here could be useful in estimating exposure and body burden to other VOCs in these three classes. Median residence times for the nine VOCs ranged from 3-12 min for compartment 1 (metabolizing); 0.3-2 h for compartment 2; 2-5 h for compartment 3; and 1-4 d for compartment 4. The fraction of the parent compound exhaled at equilibrium was estimated to range from 0.06-0.16 for four aromatic compounds and decane; 0.22-0.23 for trichloroethylene and dichloromethane; 0.35 for hexane; and 0.88 for 1,1,1-trichloroethane. Limited blood measurements were obtained for six of the nine VOCs in two subjects simultaneously with the breath samples over four-hour decay periods. Blood/breath ratios agreed well between the two subjects, but were higher than human blood/air partition coefficients reported in subjects exposed to high concentrations. This observation is consistent with results from other studies at relatively low concentrations.

Effect of dry-cleaned clothes on tetrachloroethylene levels in indoor air, personal air, and breath for residents of several New Jersey homes.

Several volatile organic compounds, including tetrachloroethylene, have been found to be nearly ubiquitous in residential indoor environments during previous TEAM studies. Eleven homes in New Jersey were monitored over three or five days to examine the effect of bringing freshly dry-cleaned clothes into the home on indoor air levels and personal exposures to tetrachloroethylene. Indoor air, personal air, and breath concentrations were measured over multiple 12-hrs periods before and after dry-cleaned clothes were introduced into nine of the homes. No dry-cleaned clothes were introduced into the two remaining homes. Outdoor air tetrachloroethylene concentrations were measured at six of the eleven homes. Indoor/outdoor concentration ratios and source strengths were calculated at the six homes with outdoor measurements. Elevated indoor air levels and human exposures to tetrachloroethylene were measured at seven of the nine homes with dry-cleaned clothes. Indoor air concentrations reached 300 micrograms/m3 in one home and elevated indoor levels persisted for at least 48 hrs in all seven homes. Indoor/outdoor tetrachloroethylene concentration ratios exceeded 100 for the four homes with both dry-cleaned clothes and outdoor measurements. Maximum source strengths ranged from 16 to 69 mg/hr in these homes and did not directly correspond to the number of dry-cleaned garments brought into the home. Breath levels of tetrachloroethylene increased two to six-fold for participants living in seven homes with increased indoor air levels. Indoor air, personal air, and breath tetrachloroethylene concentrations were significantly related (0.05 level) to the number of garments introduced divided by the home volume.

Temporal variability of benzene exposures for residents in several New Jersey homes with attached garages or tobacco smoke.

The U.S. Environmental Protection Agency's (EPA) previous TEAM studies of personal exposure to VOCs for 700 residents in several U.S. cities found that indoor air concentrations were often higher than outdoor levels. Several potential sources of benzene exposure were identified, including environmental tobacco smoke and materials or activities associated with attached garages. Indoor, personal, and outdoor monitoring was conducted at eleven New Jersey homes over multiple 12-hr monitoring periods. One study objective was to assess the impact of attached garages on human exposure to benzene and the variability of benzene exposure across time. Benzene was also measured in several homes inhabited by smokers and in homes without known combustion sources for comparative purposes. At homes with a garage or environmental tobacco smoke, mean indoor and personal benzene concentrations were two to five times higher than outdoor levels at all but one home. Mean personal exposures ranged from 8 to 31 micrograms/m3. Indoor/outdoor ratios were calculated and ranged from 0.8 to 11. Benzene levels in the four garages ranged from 3 to 196 micrograms/m3 and usually were higher than either indoor living areas or personal levels. Multi-zone air exchange rates were measured, and benzene source strengths in each zone were estimated. Garage source strength estimates for benzene ranged from 310 to 52,000 micrograms/h. The mass transfer of benzene from sources in the garage to home living areas was also large in three of the homes, ranging from 730 to 26,000 micrograms/h. Materials or activities in the garage were a source of benzene exposure for the residents in these three homes. Large temporal variations (factors of 2 to 30) were observed in indoor and personal benzene concentrations, indoor/outdoor ratios, and source strengths over the six or ten monitoring periods at each home. Changes in outdoor air benzene levels were an underlying factor in changing exposure levels, with indoor sources further elevating indoor air levels and personal exposures.

The National Human Activity Pattern Survey (NHAPS): a resource for assessing exposure to environmental pollutants.

Because human activities impact the timing, location, and degree of pollutant exposure, they play a key role in explaining exposure variation. This fact has motivated the collection of activity pattern data for their specific use in exposure assessments. The largest of these recent efforts is the National Human Activity Pattern Survey (NHAPS), a 2-year probability-based telephone survey (n=9386) of exposure-related human activities in the United States (U.S.) sponsored by the U.S. Environmental Protection Agency (EPA). The primary purpose of NHAPS was to provide comprehensive and current exposure information over broad geographical and temporal scales, particularly for use in probabilistic population exposure models. NHAPS was conducted on a virtually daily basis from late September 1992 through September 1994 by the University of Maryland's Survey Research Center using a computer-assisted telephone interview instrument (CATI) to collect 24-h retrospective diaries and answers to a number of personal and exposure-related questions from each respondent. The resulting diary records contain beginning and ending times for each distinct combination of location and activity occurring on the diary day (i.e., each microenvironment). Between 340 and 1713 respondents of all ages were interviewed in each of the 10 EPA regions across the 48 contiguous states. Interviews were completed in 63% of the households contacted. NHAPS respondents reported spending an average of 87% of their time in enclosed buildings and about 6% of their time in enclosed vehicles. These proportions are fairly constant across the various regions of the U.S. and Canada and for the California population between the late 1980s, when the California Air Resources Board (CARB) sponsored a state-wide activity pattern study, and the mid-1990s, when NHAPS was conducted. However, the number of people exposed to environmental tobacco smoke (ETS) in California seems to have decreased over the same time period, where exposure is determined by the reported time spent with a smoker. In both California and the entire nation, the most time spent exposed to ETS was reported to take place in residential locations.

The F plasmid traY gene product binds DNA as a monomer or a dimer: structural and functional implications.

The F factor traY gene product (TraYp) is a site-specific DNA-binding protein involved in initiation of DNA transfer during bacterial conjugation. The sequence of TraYp exhibits a unique direct-repeat structure predicted to have a ribbon-helix-helix DNA-binding motif in each repeat unit. The stoichiometry of TraYp binding to DNA was determined to further support the hypothesis that TraYp is a member of the ribbon-helix-helix family of DNA-binding proteins. A glutathione-S-transferase-traY fusion protein was purified and shown to possess almost wild-type DNA-binding activity. DNA-binding experiments were performed in which the DNA ligand was incubated with either the fusion protein, the wild-type protein, or both. The results indicate that TraYp can bind DNA as a monomer or a dimer. Thus a TraYp monomer folds into a stable three-dimensional structure similar to that of a dimer of the ribbon-helix-helix proteins Arc or Mnt. A homology model of a TraYp monomer has been constructed using the co-crystal structure of Arc bound to DNA as a template to provide additional support for this conclusion. In addition, we have shown that an origin of the transfer-deletion mutant lacking approximately half of the TraYp-binding site can only be bound by a monomer of TraYp. The functional implications of this result are discussed.

Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I.

DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origin of transfer (oriT) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T. L. Thompson, M. B. Centola, and R. C. Deonier, J. Mol. Biol. 207:505-512, 1989). In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction. Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitation of nicked DNA with cold KCl in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule. The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein. On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage. A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.

Stepwise assembly of a relaxosome at the F plasmid origin of transfer.

A central step in the transfer of genetic information during bacterial conjugation of the Escherichia coli F plasmid involves the formation of a protein-DNA complex, called the relaxosome, at the origin of transfer. During conjugation, the relaxosome introduces a site- and strand-specific nick from which the physical transfer of a single strand of DNA is initiated. At least two F-encoded proteins, TraIp (traI gene product) and TraYp (traY gene product), and one host-encoded protein, integration host factor, are involved in this process. In this report, we use DNase I protection and electron microscopic techniques to investigate the mechanism of relaxosome formation. Our results show that TraYp and integration host factor form a protein-DNA complex that facilitates the binding of TraIp to assemble a relaxosome capable of introducing a site- and strand-specific nick at the origin of transfer. This nick is identical to that observed during conjugation.

Characterization of the Escherichia coli F factor traY gene product and its binding sites.

The traY gene product (TraYp) from the Escherichia coli F factor has previously been purified and shown to bind a DNA fragment containing the F plasmid oriT region (E. E. Lahue and S. W. Matson, J. Bacteriol. 172:1385-1391, 1990). To determine the precise nucleotide sequence bound by TraYp, DNase I footprinting was performed. The TraYp-binding site is near, but not coincident with, the site that is nicked to initiate conjugative DNA transfer. In addition, a second TraYp binding site, which is coincident with the mRNA start site at the traYI promoter, is described. The Kd for each binding site was determined by a gel mobility shift assay. TraYp exhibits a fivefold higher affinity for the oriT binding site compared with the traYI promoter binding site. Hydrodynamic studies were performed to show that TraYp is a monomer in solution under the conditions used in DNA binding assays. Early genetic experiments implicated the traY gene product in the site- and strand-specific endonuclease activity that nicks at oriT (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980; S. McIntire and N. Willetts, Mol. Gen. Genet. 178:165-172, 1980). As this activity has recently been ascribed to helicase I, it was of interest to see whether TraYp had any effect on this reaction. Addition of TraYp to nicking reactions catalyzed by helicase I showed no effect on the rate or efficiency of oriT nicking. Roles for TraYp in conjugative DNA transfer and a possible mode of binding to DNA are discussed.