Linear plasmids of the bacterium Borrelia burgdorferi have covalently closed ends.

The genetics of spirochetes, a division of eubacteria, has been little studied. Double-stranded linear plasmids were found in Borrelia burgdorferi, the agent of Lyme disease. A 49-kilobase linear plasmid contained the ospA and ospB genes, which encode the major outer membrane proteins of strain B31. Molecules of the 49-kilobase plasmid rapidly reannealed after alkaline denaturation; rapid renaturation was prevented if the 49-kilobase plasmids were first treated with S1 nuclease. When denatured plasmid molecules were examined directly, single-stranded circles of approximately 100-kilobase circumference were seen. These studies provide direct visual evidence that the linear plasmids have covalently closed ends. This form of DNA occurs in some animal viruses, but it has not heretofore been described in prokaryotic organisms.

Spiroplasmavirus citri 3: propagation, purification, proteins, and nucleic acid.

SVC3 is a short-tailed polyhedral virus particle morphologically detectable in many spiroplasmas. It was isolated from two different spiroplasmas (Spiroplasma citri and the suckling mouse cataract agent) by infecting lawns and broth culture of another strain of Spiroplasmavirus citri. Virions from either donor strain had a buoyant density of 1.26 grams per cubic centimeter (metrizamide) or 1.45 grams per cubic centimeter (cesium chloride), and contained five proteins and linear double-stranded DNA with a molecular weight of 14 X 10(6). Other spiroplasmaviruses have not been propagated, and the molecular weights of double-stranded DNA from other mycoplasma (Acholeplasma) viruses are unknown.

Molecular cloning of polyoma virus DNA in Escherichia coli: lambda phage vector system.

The biological activity of recombinant phage and recombinant phage DNA containing monomeric or dimeric polyoma DNA inserts was examined in mice and cultured mouse cells. Recombinant preparations containing a single copy of viral DNA were invariably noninfectious; molecules containing a dimeric polyoma DNA insert were at least seven orders of magnitude less infectious than polyoma virions after parenteral inoculation. No infection was detected with any recombinant preparation after oral administration.

The structural gene for tetanus neurotoxin is on a plasmid.

A pool of synthetic oligonucleotides was prepared based on the amino terminal amino acid sequence of tetanus toxin. This probe hybridized to plasmid DNA isolated from three toxigenic strains of Clostridium tetani but not to plasmid DNA from a nontoxigenic strain. These results show that the structural gene for the toxin is on the plasmid. The pCL1 plasmid from one of the toxigenic strains spontaneously deleted 22 kilobase pairs of DNA to form pCL2. Strains harboring this deleted plasmid are nontoxigenic. However, the probe mixture hybridized to pCL2, indicating that the DNA encoding the amino terminus of the toxin had not been deleted. Restriction endonuclease cleavage maps of pCL1 and pCL2 were constructed and indicate the approximate location and orientation of the structural gene for tetanus toxin.

Molecular cloning and sequence of the concatemer junction from vaccinia virus replicative DNA. Viral nuclease cleavage sites in cruciform structures.

The concatemer junction from replicative forms of vaccinia virus DNA was cloned into plasmid vectors and shown to be a precise duplex copy of the viral terminal hairpin structure, with each strand corresponding to one of the alternative sequence isomers. The plasmids were relaxed circles with extruded cruciforms representing two copies of the vaccinia telomere hairpin structure. Head-to-head dimers containing two copies of the vaccinia virus concatemer junction were observed to contain only one set of stem-loop structures per molecule, suggesting that the initial formation of a small cruciform, and not branch migration, was the rate-limiting step in cruciform formation. The plasmids containing the concatemer junction were converted into nicked circular, linear and cross-linked linear molecules by a nuclease isolated from vaccinia virions. The region-specific cleavage near the border of the hairpin loop and the formation of DNA cross-links in some of the molecules is consistent with the nuclease acting as a nicking-closing enzyme that participates in the resolution of mature termini from replicative concatemer intermediates.

Vapor-phase and fine particulate matter concentrations of polycyclic aromatic hydrocarbons measured during the winter months in a northern Rocky Mountain urban airshed.

A fine particulate matter (PM2.5) sampling program was conducted in Missoula, MT, to investigate both the particle and vapor phases of PM2.5-associated polycyclic aromatic hydrocarbons (PAHs) found in a northern Rocky Mountain urban airshed. Twenty-four-hour samples were collected during the cold winter months of January through April 2002, when many of the more volatile organic components of PM2.5 were expected to be found in the condensed particle form. To meet analytical detection limits, each of the 12 individual sample days were aggregated into four total filter and polyurethane foam (PUF) samples, respectively, with each aggregate containing 3 sample days. Quartz filter (particle-phase PAHs) and PUF (vapor-phase PAHs) aggregates were analyzed separately for 18 individual PAHs and phenolics by gas chromatography/mass spectrometry. Results showed that 87% of the PM2.5-associated phenolics and PAHs measured in this study were found in the vapor phase. PM2.5-associated gas/particle partition coefficients (Kp,2.5) ranged from 0 for the lighter phenolics and PAHs to approximately 0.1 for some of the heavier PAHs, such as fluoranthene and pyrene. Calculating Kp,2.5 for the heaviest measured PAHs was not feasible because of low or undetectable concentrations in the vapor phases of these compounds. Phenolics and two-ringed and three-ringed PAHs were found almost exclusively in the vapor phase. Four-ringed PAHs were distributed between the particle and vapor phases, with more mass measured in the vapor phase. Very little five-ringed and higher PAHs were measured from either the filter or PUF sampling medium. These results provide information on both the concentrations and different phases of PM2.5-associated PAHs measured during the winter months in a northern Rocky Mountain urban airshed, when concentrations of PM2.5 are generally at their highest compared with the rest of the year.

The Missoula Valley semivolatile and volatile organic compound study: seasonal average concentrations.

The city of Missoula is located in a high mountain valley (elevation 3200 ft.) in western Montana and contains one of the largest populations in the entire Rocky Mountain Region completely enclosed by mountains. During the 2000/2001 Missoula Valley Sampling Program, ambient levels of 61 semivolatile organic compounds (SVOCs) and 54 volatile organic compounds (VOCs) were originally quantified before refining the analytical program to 28 of the most prominent SVOCs and VOCs found in the Missoula Valley airshed. These compounds were measured over 24-hr periods at two locations throughout an entire year. This study provides the first, comprehensive appraisal of the levels of SVOCs and VOCs measured simultaneously throughout all four seasons at two locations in the Missoula Valley, including those levels measured during the 2000 Montana wildfire season. Generally, SVOC levels were comparable between both sides of the Missoula Valley. However, there were nearly double the amount of VOCs measured at the more urban Boyd Park site compared with the rural Frenchtown sampling site, a result of the greater number of automobiles on the eastern side of the Valley. SVOCs and VOCs were measured at their highest levels of the sampling program during the winter. Forest fire smoke samples collected during the summer of 2000 showed significant increases in SVOC phenolic compounds, including phenol, 2-methylphenol, 4-methylphenol, and 2,4-dimethylphenol. Although there were modest increases in some of the other SVOCs and VOCs measured during the fire season, none of the increases were as dramatic as the phenolics.

Cloning and expression of the Borrelia burgdorferi lon gene.

The ATP-dependent protease Lon (La) of Escherichia coli degrades abnormal proteins and is involved in the regulation of capsular polysaccharide synthesis. In addition, mutations in the E. coli lon gene suppress temperature-sensitive mutations in other genes. The lon gene of Borrelia burgdorferi, encoding a homolog of the Lon protease, has been cloned and sequenced. The gene encodes a protein of 806 amino acids. The deduced amino acid sequence of the B. burgdorferi Lon protease shares substantial sequence identity with those of other known Lon proteases. The transcription start point of the B. burgdorferi lon gene was identified by primer extension analysis and the potential promoter did not show similarities to the consensus heat-shock promoter in E. coli. The 5'-end of the B. burgdorferi lon gene appears to suppress the temperature-sensitive phenotype of an E. coli lpxA mutant.

Extrachromosomal elements of spirochetes.

The spirochetes include some important pathogenic bacteria, Treponema, Borrelia and Leptospira. The pathogeneses of these spirochetes are very diverse. In an attempt to learn more about the virulence factors among the spirochetes, their genetic organization and capacity have been studied. Structural analysis of the genome in Borrelia has shown that the genome is composed of one linear maxi-chromosome with additional linear minichromosomes as well as several supercoiled circular plasmids. Moreover, the molecular analysis of the terminal ends of one of the linear minichromosomes has revealed that this unique replicon has sequence similarities with poxviruses and particularly the viral agent of African swine fever. The presence of nucleic-acid-containing vesicles and its possible role in mediating DNA transfer between borreliae is an additional, very interesting feature of these organisms. Treponema does not contain any linear DNA, chromosomal or extrachromosomal, however molecular characterization of a 2.6-kb plasmid of Treponema denticola has been performed with the aim of establishing cloning vehicles to study the virulence properties of the genus Treponema.

A cryptic 19-kilobase plasmid associated with U.S. isolates of Yersinia pestis: a dimer of the 9.5-kilobase plasmid.

Yersinia pestis, the etiologic agent of plague, carries three prototypic plasmids with sizes of 110 kb (pFra, pTox), 70 kb (pLcr, pVW, pCad), and 9.5 kb (pPla, pPst). Studies suggest that geographic isolates of Y. pestis may be differentiated by plasmid profiles. Yersinia pestis isolated from the western United States harbor an additional plasmid, estimated to be approximately 19 kb in size. This cryptic plasmid was characterized by restriction endonuclease digestion, amplification and sequencing of the plasminogen activator gene segment, Southern blotting, and visualized by electron microscopy. Results revealed that this cryptic plasmid is a supercoiled DNA plasmid, 18.85+/-0.59 (mean+/-SD) kb in length, and is a dimer of the 9.5-kb plasmid. The genetic reason for the appearance of this form of the 9.5-kb plasmid in Y. pestis from Arizona, California, Colorado, New Mexico, and Texas is under study.

Variation in nectar volume and composition of Impatiens capensis at the individual, plant, and population levels.

Although the volume and chemical composition of nectars are known to vary among plant species and to affect pollinator response to plants, relatively little is known of the variation in volume, and sugar and amino acid composition within species. We collected nectar from Impatiens capensis in a nested design: three flowers from each of three plants from each of three populations. This design enabled us to quantify variation within individual plants, among plants within populations, and among populations. Using high performance liquid chromatography, we analyzed the sugar and amino composition of the 27 flowers. Analysis of variance showed that none of the parameters (volume, concentrations of three sugars and 24 amino compounds) varied within individuals. Variation in nectar volume was not significant among plants but was nearly significant among populations. Of the three sugars detected (sucrose, glucose, and fructose), the only significant variation was that of sucrose among populations. Concentrations of 12 amino compounds varied significantly at the plant level while 7 amino compounds varied among populations. The results indicate that: (1) pooling of nectar samples from flowers of individual plants can be an acceptable methodology for those seeking to understand within species variation; (2) amino compounds appear to vary more than either volumes or sugar concentrations; (3) future studies should assess how much of the observed variation is due to genetic versus environmental differences; (4) additional studies should examine the geographic variation in nectar parameters and pollinators of I. capensis in order to assess the role different pollinators play in shaping nectar composition.

Specific and nonspecific responses of murine B cells to membrane blebs of Borrelia burgdorferi.

Lymphocyte blastogenesis assays and immunoblotting were used to investigate and compare murine B-cell responses to preparations of extracellular membrane blebs (BAg) and spirochetes (Ag) of Borrelia burgdorferi. Immunoblotting BAg, Ag, and medium control preparations with serum from naive and infected C57BL/10 mice revealed that BAg and Ag had similar specific reactivity profiles except that major antigens of 83, 60, and 41 kDa were detected in Ag but not in BAg. It was determined that 1 microgram (dry weight) of Ag contained 0.0051 and 0.0063 microgram of outer surface proteins A (OspA) and OspB, respectively, whereas 1 microgram (dry weight) of BAg contained 0.0024 microgram of OspA and 0.0015 microgram of OspB. Both BAg and Ag caused blastogenesis in cultures of spleen cells from both groups of mice, but BAg-stimulated lymphocytes exhibited significantly greater (P < or = 0.05) blastogenesis after 2 or 6 days of culture than did lymphocytes stimulated by Ag or medium control. Flow cytometry and antibody capture enzyme-linked immunosorbent assays identified responding lymphocytes as B cells which secreted polyclonal immunoglobulin M (IgM) but not IgG or IgA. Treatment of BAg and lipopolysaccharide controls with polymyxin B resulted in as much as 20.7 and 54.3% mean decreases in blastogenesis, respectively. Fractionation of BAg or Ag by ultracentrifugation before culture with spleen cells from naive mice indicated that B-cell blastogenesis was probably associated with spirochetal membranes. The results of this study demonstrate that specific humoral responses are directed towards extracellular membrane blebs which lack the 83-, 60-, and 41-kDa antigens of intact spirochetes and that blebs also possess significant nonspecific mitogenic activity for murine B cells. This activity was not due entirely to typical lipopolysaccharide or OspA and OspB lipoproteins.