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We have found the nuclear stain DRAQ5 to decrease the cellular uptake of a series of boron dipyrromethane (bodipy)-labelled compounds. This phenomenon is consistent between Lysotracker Green DND 26, Lysotracker Red DND 99 and bodipy-labelled mycolactone. Although DRAQ5 uptake was not prevented, DRAQ5 was in significant excess in each case. As the effect is consistent among two cell types, RAW 264.7 monocyte/macrophages and Bend 3 endothelial cells, we hypothesize that it may be a result of the two dyes complexing in solution into a form that is not taken up by the cells. This hypothesis is confirmed by fluorescence resonance energy transfer analysis in solution.

Borrelia burgdorferi regulates genes in response to a number of environmental signals such as temperature and pH. A green fluorescent protein (GFP) reporter system using the ospC, ospA and flaB promoters from B. burgdorferi B31 was introduced into infectious clonal isolates of strains B31 and N40 to monitor and compare gene expression in response to pH and temperature in vitro. GFP could be assayed by epifluorescence microscopy, immunoblotting or spectrofluorometry and was an accurate reporter of target gene expression. It was determined that only 179 bp 5' of ospC was sufficient to regulate the reporter gfp in vitro in response to pH and temperature in B. burgdorferi B31. The loss of linear plasmid (lp) 25, lp28-1, lp36 and lp56 had no effect on the ability of B. burgdorferi B31 to regulate ospC in response to pH or temperature. The amount of OspC in N40 transformants was unaffected by changes in pH or temperature of the culture medium. This suggests that regulation of gene expression in response to pH and temperature may vary between these two B. burgdorferi strains.

When Borrelia burgdorferi is transmitted from the tick vector to the mammalian host, the bacterium experiences alterations in its environment, such as changes in temperature and pH. Previously, we observed numerous alterations in the membrane protein profile when B. burgdorferi B31 was grown at pH 7.0 compared to pH 8.0. Here we identify 11 genes localizing to linear plasmids that are up-regulated at pH 7.0 relative to pH 8.0 in vitro. Seven genes (bba03, bba24, bba64, bba66, bbe31, bbj41/bbi39 [encoding products that are 99% identical], and bbk01) were indirectly identified by proteomic analysis of membrane proteins. Another gene, bba36, was identified by screening a B. burgdorferi B31 genomic library with cross-adsorbed hyperimmune rabbit serum. Two additional genes, bba65 and bba73, were identified by Northern blot analysis. Genes bba64, bba65, bba66, bbj41/bbi39, and bba73 are members of paralogous gene family 54, and bbe31 is a member of the closely related paralogous gene family 60. Gene bba24 is part of a bicistronic operon with bba25 that encodes the well-characterized decorin binding proteins A and B. All 11 genes were transcriptionally regulated, yet the degree of pH regulation varied, with some genes more tightly regulated than others. The regions upstream of these pH-regulated genes appeared to be unrelated, yet many contained dyad repeats ranging from 12 to 25 nucleotides in length that may be involved in the regulation of these genes.

Historically, a number of bacteriophage-like particles have been observed in association with members of the bacterial order Spirochetales, the spirochetes. In the last decade, several spirochete bacteriophages have been isolated and characterized at the molecular level. We have recently characterized a bacteriophage of the Lyme disease agent, Borrelia burgdorferi, which we have designated phiBB-1. Here we review the history of the association between the spirochetes and their bacteriophages, with a particular emphasis on phiBB-1 and its prophage, the 32-kb circular plasmid family of B. burgdorferi.

The mosquito Aedes aegypti is capable of transmitting a variety of pathogens to man and to other vertebrates. The midgut of this insect has been well-studied both as the tissue where the first contact occurs between ingested pathogens and the insect host, and as a model system for blood meal digestion in blood-sucking insects. To understand better the nature of the midgut surface encountered by parasites or viruses, we used scanning electron microscopy to identify the most prominent structures and cell morphologies on the luminal midgut surface. The luminal side of the midgut is a complex and layered set of structures. The microvilli that are found on most, but not all, cells are covered by a network of fine strands that we have termed the microvilli-associated network (MN). The MN strands are membranous, as shown by a membrane bilayer visible in cross sections of MN strands at high magnification in transmission electron micrographs. The MN is found in blood-fed as well as unfed mosquitoes and is not affected by chitinase treatment, suggesting that it is not related to the chitinous peritrophic membrane that is formed only after blood feeding. The cells in the midgut epithelium have two distinct morphologies: the predominant cell type is densely covered with microvilli, while cells with fewer microvilli are found interspersed throughout the midgut. We used lectins to probe for the presence of carbohydrates on the midgut surface. A large number of lectins bind to the luminal midgut surface, suggesting that a variety of sugar linkages are present on the structures visualized by electron microscopy. Some of these lectins partially block attachment of malaria ookinetes to the midgut surface in vitro. Thus, the mosquito midgut epithelium, like the lining of mammalian intestines, is complex, composed of a variety of cell types and extensively covered with surface carbohydrate that may play a role in pathogen attachment.

Borrelia burgdorferi, the causative agent of Lyme disease, alternates between the microenvironments of the tick vector, Ixodes scapularis, and a mammalian host. The environmental conditions the spirochete encounters during its infectious cycle are suspected to differ greatly in many aspects, including available nutrients, temperature, and pH. Here we identify alterations in the membrane protein profile, as determined by immunoblotting and two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), that occur in virulent B. burgdorferi B31 as the pH of the medium is altered. Initial comparisons of cultures incubated at pHs 6.0, 7.0, and 8.0 yielded alterations in the expression of seven membrane proteins as determined by probing with hyperimmune rabbit serum. Six of these membrane proteins (54, 45, 44, 43, 35, and 24 kDa) were either present in increased amounts in or solely expressed by cultures incubated at pHs 6.0 and 7.0. The 24-kDa protein that decreased in expression at pH 8.0 was identified as outer surface protein C (OspC). In addition, a 42-kDa membrane protein increased in amount in cultures incubated at pH 8.0. Similar changes were observed with serum from a mouse infected by tick bite, with the recognition of two additional bands (48 and 46 kDa) unique to pHs 6. 0 and 7.0. When membrane fractions were analyzed by 2D-NEPHGE, at least 37 changes in the membrane protein profile between cells incubated at pHs 6.0, 7.0, and 8.0 were observed by immunoblotting and silver staining. Environmental cues such as pH may prove important in the regulation of virulence determinants and factors necessary for the adaptation of B. burgdorferi to the tick or mammalian microcosm.

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.

Plasmodium gallinaceum ookinetes adhered to Aedes aegypti midgut epithelia when purified ookinetes and isolated midguts were combined in vitro. Ookinetes preferentially bound to the microvillated luminal surface of the midgut, and they seemed to interact with three types of structures on the midgut surface. First, they adhered to and migrated through a network-like matrix, which we have termed microvilli-associated network, that covers the surface of the microvilli. This network forms on the luminal midgut surface in response to blood or protein meals. Second, the ookinetes bound directly to the microvilli on the surface of the midgut and were occasionally found immersed in the thick microvillar layer. Third, the ookinetes associated with accumulations of vesicular structures found interspersed between the microvillated cells of the midgut. The origin of these vesicular structures is unknown, but they correlated with the surface of midgut cells invaded by ookinetes as observed by TEM. After binding to the midgut, ookinetes underwent extensive morphological changes: they frequently developed one or more annular constrictions, and their surface roughened considerably, suggesting that midgut components remain bound to the parasite surface. Our observations suggest that, in a natural infection, the ookinete interacts in a sequential manner with specific components of the midgut surface. Initial binding to the midgut surface may activate the ookinete and cause morphological changes in preparation for invasion of the midgut cells.

The emergence and rapid rise to dominance of Vibrio cholerae O139 in India and Bangladesh in 1992 led to the consideration that choleraphage might serve as both a selective mechanism and a means for horizontal transmission of genetic information. A filamentous phage '493' from O139 strain AJ27-493 has been purified and partially characterized. The phage was inactive on classical biotype V. cholerae O1 but it was active on El Tor biotype strains isolated prior to 1994 when El Tor re-emerged in Bangladesh. More recent El Tor isolates were all resistant to the phage. The phage was also active on O139 strains. Unlike the filamentous ctx phi, the receptor for 493 is not TcpA. The phage genome was a 9.3 kb closed circular single-stranded molecule containing a 0.4 kb double-stranded stem supporting a 2 kb single-stranded loop. A 283 bp fragment was cloned and used as a probe in Southern hybridization, in parallel with total phage 493 DNA. These probes hybridized both chromosomally and extrachromosomally with most O139 strains, but not with O1 strains. Infection of hybridization-negative El Tor or O139 strains resulted in the presence of hybridizing loci (both plasmid and chromosomal), in the appearance of an 18 kDa protein, and in marked alterations in colonial morphology. Phage 493 is clearly distinct from other O139 choleraphages which have been described. Phage 493 DNA hybridized with an encapsulated non-O1 (O31) strain (NRT36S) which was isolated before O139 was recognized. NRT36S also produces a phage which can infect El Tor strains with low efficiency. Further studies may reveal whether bacteriophage play a role in the emergence and the territoriality of new choleragenic vibrios.

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.

Borrelia burgdorferi has demonstrated a capacity to resist the in vitro effects of powerful eukaryotic and prokaryotic metabolic inhibitors. However, treatment of laboratory cultures on Barbour-Stoenner-Kelly medium with melittin, a 26-amino acid peptide contained in honeybee venom, showed immediate and profound inhibitory effects when they were monitored by dark-field microscopy, field emission scanning electron microscopy, and optical density measurements. Furthermore, at melittin concentrations as low as 100 microg/mL, virtually all spirochete motility ceased within seconds of inhibitor addition. Ultrastructural examination of these spirochetes by scanning electron microscopy revealed obvious alterations in the surface envelope of the spirochetes. The extraordinary sensitivity of B. burgdorferi to mellitin may provide both a research reagent useful in the study of selective permeability in microorganisms and important clues to the development of effective new drugs against lyme disease.

We have used short oligonucleotides to genetically transform the Lyme disease spirochaete Borrelia burgdorferi. The oligonucleotides are derived from the sequence of an Arg-133 to Ile mutant gyrB (chromosomal) gene that confers resistance to the antibiotic coumermycin A1. Oligonucleotides were about 10,000-fold less efficient at transformation, on a molar basis, than longer PCR-generated substrates. All of the transformants tested contained the predicted site-directed silent mutation in their gyrB genes. Antisense oligonucleotides were more efficient at transformation than either sense or double-stranded oligonucleotides. This is the first demonstration of oligonucleotides used to introduce site-directed mutations directly into the genome of a bacterium.

The polysialic acid (polySia) capsule of Escherichia coli K1 is a key virulence determinant of the organism, allowing it to evade host defenses. The proteins necessary for expression of the capsule are encoded by the 17 kb kps gene cluster. This cluster contains two genes, kpsM and kpsT, that are required for polySia transport across the cytoplasmic membrane. KpsM is a hydrophobic integral inner membrane protein, while KpsT is a peripheral inner membrane protein that binds ATP. They belong to the ATP-binding cassette (ABC) superfamily of transporters. To study the role of KpsT in polySia translocation, we used PCR mutagenesis to isolate dominant negative mutations of plasmid-encoded kpsT. All mutations mapped to the same glutamic acid residue at position 150, adjacent to Walker motif B of KpsT. Wild-type (kps+) cells harboring one such allele, E150G, did not transport polySia to the cell surface but accumulated intracellular polysaccharide and produced small colonies containing cells that grew as long filaments. The E150G protein still bound ATP as shown by 8-azidoATP photolabeling assays. We combined the E150G allele with each of five mutations isolated previously in kpsT. Mutations that disrupt ATP-binding (K44E) or alter regions of the protein thought to interact with KpsM (G84D, S126F) suppressed the dominant negative phenotype while mutations in the C-terminal portion of the protein (C163Y, H181Y) did not suppress. These studies have allowed the development of a working model for the role of KpsT in polySia chain translocation.

No useful method to genetically manipulate Borrelia burgdorferi, the causative agent of Lyme disease, has been developed previously. We have used resistance to the coumarin antibiotic coumermycin A1, an inhibitor of DNA gyrase, as a genetic marker to monitor the transformation of B. burgdorferi by electroporation. Introduction of site-directed mutations into the gyrB gene demonstrated that transformation was successful, provided evidence that homologous recombination occurs on the chromosome, and established that mutations at Arg-133 of DNA gyrase B confer coumermycin A1 resistance in B. burgdorferi. The coumermycin A1-resistant gyrB marker and genetic transformation can now be applied toward dissecting the physiology and pathogenesis of the Lyme disease agent on a molecular genetic level.

Analysis of the ospD gene has revealed that this gene is not universal among Lyme disease spirochete isolates. The gene was found to be carried by 90, 50, and 24% of the Borrelia garinii, B. afzelii, and B. burgdorferi isolates tested. Size variability in the ospD-encoding plasmid was also observed. Sequence analysis has demonstrated the presence of various numbers of a 17-bp repeated sequence in the upstream control (promoter) region of the gene. In addition, a region within the coding sequence where various insertions, deletions, and direct repeats occur was identified. ospD gene sequences from 31 different isolates were determined and utilized in pairwise sequence comparisons and construction of a gene tree. These analyses suggest that the ospD gene was the target of several recombinational events and that the gene was recently acquired by Lyme disease spirochetes and laterally transferred between species.

We have isolated and characterized mutants of Borrelia burgdorferi that are resistant to the antibiotic coumermycin A1, which targets the B subunit of DNA gyrase. Mutants had either 100- or 300-fold higher resistance to coumermycin A1 than wild-type B. burgdorferi. In each case, a single point mutation in the gyrB gene converted Arg-133 to Gly or Ile. Mutations in the homologous Arg residue of Escherichia coli DNA gyrase are also associated with resistance to coumarin antimicrobial agents.

The aetiological agents of Lyme disease form a phylogenetically heterogeneous group, composed of three species, Borrelia burgdorferi, Borrelia garinii, and group VS461. We have compared the sizes of the linear plasmid that carries the genes encoding the major outer-surface proteins OspA and OspB as well as the size and structure of the chromosome among the Lyme disease spirochaetes. We have found differences in the sizes of the ospA-containing plasmids, but not the linear chromosomes among the three species. The ospA-containing plasmid size of 50 kb in B. burgdorferi isolates is significantly smaller than the size of 55 kb in B. garinii isolates and 56 kb in group VS461 isolates. The chromosome was found to be linear in all three Borrelia species, but not significantly different in size.

Using oligonucleotide probes which have previously been shown to be specific for the ospC gene found in the Lyme disease spirochete species Borrelia burgdorferi, B. garinii, and group VS461, we detected an ospC homolog in other Borrelia species including B. coriaceae, B. hermsii, B. anserina, B. turicatae, and B. parkeri. In contrast to the Lyme disease spirochetes, which carry the ospC gene on a 26-kb circular plasmid, we mapped the gene in other Borrelia species to linear plasmids which varied in size among the isolates tested. Some isolates carry multiple copies of the gene residing on linear plasmids of different sizes. The analyses conducted here also demonstrate that these Borrelia species contain a linear chromosome. Northern (RNA) blot analyses demonstrated that the gene is transcriptionally expressed in all species examined. High levels of transcriptional expression were observed in some B. hermsii isolates. Transcriptional start site analyses revealed that the length of the untranslated leader sequence was identical to that observed in the Lyme disease spirochete species. By Western blotting (immunoblotting) with antiserum (polyclonal) raised against the OspC protein of B. burgdorferi, we detected an immunoreactive protein of the same molecular weight as the OspC found in Lyme disease spirochete species. The results presented here demonstrate the presence of a protein that is genetically and antigenically related to OspC which is expressed in all species of the genus Borrelia tested.

A comparison of the osp operon in 24 Lyme disease isolates, including representatives from each of the three established species, Borrelia burgdorferi, Borrelia garinii, and group VS461, was conducted. Several properties were assessed to determine whether the variability observed in this operon was reflective of the species of the isolate. At the transcriptional level, start site and Northern (RNA) blot analyses were conducted. B. garinii and VS461 group isolates were found to possess an untranslated leader sequence 6 nucleotides longer than that observed in B. burgdorferi isolates. By Northern blot analyses all Lyme disease isolates, except the B. garinii isolate VS102, were found to produce a polycistronic full-length ospAB message. Isolate VS102 produced a truncated message lacking the ospB portion of the transcript. Southern blot analyses suggest that the deletion occurred at the DNA level and was not due to a posttranscriptional event. Analysis of the outer surface proteins by two-dimensional gel electrophoresis demonstrated that the OspB isoelectric points were variable, with the OspB of B. garinii isolates exhibiting a pronounced acidic shift. The reactivity of different isolates to OspA and -B monoclonal antibodies and to a hyperimmune anti-ospAB serum was also variable. The results presented here demonstrate genotypic and phenotypic heterogeneity in the osp operon at both the inter- and intraspecies levels. The results have implications concerning the use of the osp genes or their gene products in the development of a Lyme disease vaccine, as diagnostic markers of Lyme disease, and in subtyping of Lyme disease isolates.

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.

In Lyme disease spirochetes, the ospC gene encodes a 22.7-kDa protein referred to as either the pC or the OspC protein. Using a variety of electrophoretic approaches followed by Southern blotting and probing with oligonucleotide probes, we mapped the ospC gene to a circular 26-kb plasmid. The ospC gene represents the first gene to be mapped to a circular plasmid in Lyme disease spirochetes. The occurrence of this gene in isolates belonging to each of the three Lyme disease-associated species, Borrelia burgdorferi, Borrelia garinii, and the VS461 group, was evaluated. The ospC gene was found to occur in all 21 isolates tested from each of the three species. Differential hybridization with a series of ospC probes in both Northern (RNA) and Southern blot analyses demonstrated that there is sequence variability in the ospC gene among isolates. While the gene was found to be present in all isolates, not all actively transcribed the gene. Transcriptional start site analyses suggest that the gene may be under the control of multiple promoters that are highly similar in nucleotide sequence.

Coumermycin A1 is an inhibitor of DNA gyrase, an enzyme that catalyzes supercoiling of DNA and is required for bacterial DNA replication. We have investigated the activity of this coumarin antibiotic on Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease. B. burgdorferi was more susceptible than many other eubacteria to coumermycin as well as novobiocin, another coumarin antibiotic; this contrasted with its relative resistance to the DNA gyrase inhibitors nalidixic acid, oxolinic acid, and ciprofloxacin. Coumermycin at 0.2 micrograms/ml inhibited the growth of B. burgdorferi B31 in BSK II medium. A 100-fold-lower concentration induced the relaxation of two negatively supercoiled circular plasmids within 2 h. Plasmid supercoiling was restored within 2 h of removal of coumermycin. These results suggest that B. burgdorferi has a DNA gyrase and that this enzyme's activity is required for growth. Furthermore, structural analogs of coumermycin may be considered as treatments for Lyme disease.

We have determined and compared partial 16S rRNA sequences from 23 Lyme disease spirochete isolates and aligned these with 8 sequences previously presented. The 16S rRNA signature nucleotide compositions were defined for each isolate and compared with the genomic species signature nucleotide sets previously established. To identify positions truly indicative of species classification which could serve as targets for polymerase chain reaction species-specific identification primers, 16S rRNA-based phylogenetic analyses were conducted. On the basis of the identified signature nucleotides, we designed polymerase chain reaction primer sets which (i) amplify all spirochete species associated with Lyme disease and (ii) differentiate between these species. The primer sets were tested on 38 Borrelia isolates associated with Lyme disease and were found to be sensitive and specific. All Lyme disease isolates tested were amplification positive. These primers allow for the rapid species identification of Lyme disease isolates.

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.

As part of a continuing effort to assess genetic variation among isolates of Borrelia burgdorferi we have determined the 16S rRNA signature nucleotide makeup of two tick isolates from the USSR. Signature nucleotides were identified via reverse transcriptase primer extension sequencing of select regions of the 16S rRNA molecule. In addition, the near complete 16S rRNA sequence of one of the isolates, R-IP3, was determined and utilized in a phylogenetic assessment. The sequence was aligned with the 16S rRNA sequences of other B. burgdorferi isolates as well as with other Borrelia species. Distance matrix analyses were performed and a phylogenetic tree was constructed. These analyses demonstrate that these isolates belong to a third previously unidentified genomic group of B. burgdorferi.

Examination of a number of previously published aligned Borrelia 16S rRNA sequences revealed the presence of regions which could serve as oligonucleotide probe targets for both species-specific identification of Borrelia burgdorferi and distinction between genomic groups. Total cellular RNA isolated from Borrelia cultures was used in slot blot analysis. Radiolabeled oligonucleotides designed to hybridize to specific 16S rRNA targets were used as probes. These probes allowed for both species-specific identification and genomic group typing of B. burgdorferi.

Previous work showed that outer surface protein A (OspA) and OspB of Borrelia burgdorferi may occur within an extracellular multiprotein complex, which was resolved by electrophoresis as an 83-kDa major extracellular protein band. To characterize the 83-kDa band, we sequenced the N terminus of the predominant peptide in the band and examined the interaction between the associated proteins. Peptide sequence and amino acid composition comparisons showed identity with the heavy chain of immunoglobulin M (IgM). Reduction sensitivity experiments and the recognition of the band by antibodies specific for rabbit mu chain indicated that the multiprotein complex contained pentameric IgM. Immunoelectron microscopy showed that anti-mu chain antibodies and monoclonal antibodies to OspA and OspB bound to extracellular amorphous material surrounding cells. Furthermore, the Osps coprecipitated with either nonspecific polyclonal rabbit IgM antibodies or with murine monoclonal anti-human serum albumin IgM antibodies, using insoluble anti-mu chain antibody conjugates. Although the apparent 83-kDa complex was stable under conditions of chelation and concentrated salts, it was disrupted by treatment with neuraminidase. These results indicate that extracellular B. burgdorferi proteins, including OspA and OspB, interact with IgM. The association is apparently not a classic antibody-antigen interaction but may result from other mechanisms.

We have sequenced the 16S rRNA molecules from four species of Borrelia and from six isolates of Borrelia burgdorferi via the reverse transcriptase primer extension method. The sequences were aligned and evolutionary relationships were determined, including the calculation of evolutionary distances and the construction of a phylogenetic tree. These analyses demonstrate significant divergence among B. burgdorferi isolates, with the European isolates G1 and G2 residing most distant from the main cluster. Signature nucleotides which distinguish B. burgdorferi from all other members of this genus and which distinguish the European isolates G1 and G2 from the North American isolates B31, Sh-2-82, and 1352 were identified. Finally, Southern blot analyses were performed to compare the restriction patterns of the genes coding for rRNA and to relate our data to the grouping scheme of Postic et al. (D. Postic, C. Edlinger, C. Richaud, F. Grimont, J. Dufresne, P. Perolat, G. Baranton, and P. A. D. Grimont, Res. Microbiol. 141:465-475, 1990).

Current biological and serological techniques for demonstrating infections by Borrelia burgdorferi can be inconclusive. In order to monitor Lyme borreliosis, we developed a rapid and sensitive assay for B. burgdorferi antigens in infected hosts. Polyclonal rabbit antisera were raised against membrane vesicles and an 83-kDa vesicle-associated protein band that was purified from in vitro B. burgdorferi cultures. Immunoglobulin G (IgG) antibodies were recovered from these sera and tested for a species-specific reaction with several geographically diverse Borrelia isolates by immunoblot analysis. Parlodion-coated electron microscope grids were activated with anti-vesicle F(ab')2 fragments and then incubated with confirmed or experimental sources of spirochetal antigens. Such sources included cultured spirochetes; spirochete culture supernatants; samples of urine, blood, or serum from mice, dogs, and humans; triturates of Ixodes ticks; and bladder, spleen, liver, kidney, heart, or brain tissues from infected or control mice. Captured antigens were assayed by immune electron microscopy by using anti-83-kDa IgG antibodies and protein A-colloidal gold conjugates. The results indicated that B. burgdorferi appears to shed surface antigens which are readily detectable in urine, blood, and several organs from infected hosts. Such antigens were detectable in mouse urine at dilutions exceeding 10(-6). Intact spirochetes were frequently observed on grids incubated with blood, spleen, or bladder preparations, and B. burgdorferi was reisolated from the urinary bladders of all experimentally infected mice. These results indicated that B. burgdorferi antigens arise in a variety of host materials. Such antigens can be captured and identified with specific polyclonal antibodies, providing a sensitive assay for monitoring and studying Lyme borreliosis.

Structural analysis by electronmicroscopy and pulsed field agarose gel electrophoresis of the genome of Borrelia burgdorferi has revealed a 1000 kilobase (kb) chromosome and a unique type of extra chromosomal linear DNA, as well as supercoiled circular DNA. The plasmid DNA ranged in size from 1.5 to 50 kb. Gene mapping of the ospA and ospB genes have placed them on the 49 kb linear plasmid molecule. The osp operon of B. burgdorferi has been extensively characterized at the molecular level.

The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by variable antigens, Vmp proteins, in the outer membrane. Through nonreciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene downstream from a common expression site. To further characterize this activating site, we determined the nucleotide sequence of 6.9 kb of the common upstream expression region of strain HS1 of B. hermsii. Preceding the vmp gene promoter and a poly(dT.dA) run were three imperfectly repeated segments of 2 kb. Each of the 2-kb segments contained 1-kb elements with inverted repeats of approximately 0.2 kb each at their termini. The potential of the 1-kb elements to form stem-and-loop structures was demonstrated by heteroduplex analysis. There was no evidence of the presence of the elements elsewhere in the genome of B. hermsii. One or more of these elements may confer the unidirectionality that characterizes vmp gene switches.

The 5'-terminal palindrome of the ADV-G strain of Aleutian mink disease parvovirus (ADV) was molecularly cloned and sequenced. A full-length molecular clone of ADV-G, denoted pXVB, was then constructed. When this clone was transfected into cell cultures, infectious ADV could be rescued. Virus derived from pXVB was nonpathogenic for adult mink, as is the parent ADV-G strain.

Recently, DNA packaged within nuclease-resistant membrane vesicles of Neisseria gonorrhoeae and Borrelia burgdorferi was described. This study assayed 18 species of gram-negative and gram-positive eubacteria for nuclease-protected DNA associated with extracellular membrane vesicles. Vesicles from only the gram-negative bacteria contained nuclease-protected linear or supercoiled DNAs or both.

The wild mouse ecotropic retrovirus (WM-E) induces a spongiform neurodegenerative disease in mice after a variable incubation period of 2 months to as long as 1 year. We isolated a molecular clone of WM-E (15-1) which was weakly neurovirulent (incidence, 8%) but was highly leukemogenic (incidence, 45%). Both lymphoid and granulocytic leukemias were observed, and these leukemias were often neuroinvasive. A chimeric virus was constructed containing the env and 3' pol sequences of 15-1 and long terminal repeat (LTR), gag, and 5' pol sequences from a clone of Friend murine leukemia virus (FB29). FB29 has been shown previously to replicate to high levels in the central nervous system (CNS) but is not itself neurovirulent. This finding was confirmed at the DNA level in the current study. Surprisingly, intraperitoneal inoculation of neonatal IRW mice with the chimeric virus (FrCasE) caused an accelerated neurodegenerative disease with an incubation period of only 16 days and was uniformly fatal by 23 days postinoculation. Introduction of the LTR of 15-1 into the FrCasE genome yielded a virus (FrCasEL) with a degree of neurovirulence intermediate between those of 15-1 and FrCasE. No differences were found in the levels of viremia or the relative levels of viral DNA in the spleens of mice inoculated with 15-1, FrCasE, or FrCasEL. However, the levels of viral DNA in the CNS correlated with the relative degrees of neurovirulence of the respective viruses (FrCasE greater than FrCasEL greater than 15-1). Thus, the env and 3' pol sequences of WM-E (15-1) were required for neurovirulence, but elements within the LTR and gag-pol regions of FB29 had a profound influence on the level of CNS infection and the rate of development of neurodegeneration.

Borrelia burgdorferi, the causative agent of Lyme borreliosis, contains linear and supercoiled circular (SC) plasmids. Because SC plasmids are present in multiple copies, these plasmids were examined for species-specific sequences that could serve as high-copy-number target DNAs for a diagnostic probe. Three EcoRI fragments (4.3, 4.2, and 3.5 kilobase pairs [kb]) that hybridized with multiple DNA fragments from B. burgdorferi were identified and cloned from a SC plasmid-enriched fraction. The 4.2- and 3.5-kb fragments were similar in that they hybridized with each other and with similar-sized EcoRI fragments from two unrelated strains of B. burgdorferi. The 4.3-kb fragment did not hybridize with the other two cloned sequences. Both types of sequences hybridized with most of the SC plasmids in seven B. burgdorferi isolates, whereas only a single 49-kb linear plasmid, found in two of the seven strains tested, hybridized with the cloned sequences. None of the cloned sequences hybridized with chromosomal DNA from B. burgdorferi or with total DNA or SC plasmids from Borrelia hermsii, B. turicatae, B. coriaceae, B. parkeri, or B. anserina. These data indicate that the repeated DNA sequences described in this study appear to be plasmid associated and specific to B. burgdorferi. Heteroduplexes formed from the 4.2- and 3.5-kb fragments showed that hybridizing regions in each fragment comprise a 1.8-kb conserved region that is adjacent to a 1.5-kb region that exhibits greater sequence variability. The sequence divergence seen in the variable region is likely the result of genetic drift and may mean that these regions represent closely related genes that encode functionally similar but antigenically distinct proteins.

Linear plasmids are widely distributed in isolates of Borrelia burgdorferi, but the prevalence of supercoiled circular (SC) plasmids has not been clearly established. Enriching for SC plasmids on ethidium bromide density-gradients revealed that a low passage of strain Sh-2-82 of B. burgdorferi has at least six SC plasmids (8.4, 8.8, 20, 26, 29 and 51 kb). In addition, several of the SC plasmids were observed by electron microscopy to form concatenated structures. Because of the high incidence of coupled SC plasmids and the effect of such structures on plasmid mobility, we suggest that they could be mistaken for a single DNA species having a high molecular weight when total DNA from B. burgdorferi is examined by agarose-gel electrophoresis. Of the six SC plasmids in strain Sh-2-82, four could still be detected after two years of continuous in vitro passaging in BSK II medium. The two SC plasmids lost after a maximum of 20 passages, pBBC1 (8.4 kb) and pBBC2 (8.8 kb), shared a high degree of DNA sequence similarity, suggesting that their apparent instability may be a unique feature of these closely related plasmids. Two higher passaged variants of strain Sh-2-82, P20 and P202, were shown by hybridization to lack pBBC1 and pBBC2 sequences and were unable to infect the white-footed mouse, Peromyscus leucopus. However, six of 14 unrelated isolates that infected this rodent and Syrian hamsters also lacked pBBC1 and pBBC2 sequences. None of five unrelated but highly passaged non-infectious isolates carried either pBBC1 or pBBC2 sequences. These data indicate that pBBC1 and pBBC2 do not encode constitutively expressed proteins required for infectivity in our experimental system. Nevertheless, eight of the 13 infectious strains examined did carry pBBC1 and/or pBBC2, indicating that these two closely related plasmids can be detected in a wide range of unrelated isolates of B. burgdorferi.

Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo and transforms fibroblasts and hematopoietic target cells in vitro. We have used recombinant DNA technology to isolate and to characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by Southern blotting of restriction endonuclease digests and by electron microscopic visualization of R loops formed between the cloned DNA and MC29 or helper virus RNA. It was found that the 9.2-kilobase cloned DNA insert contains approximately 4 kilobases of viral sequences and 5.2 kilobases of quail cellular sequences. The viral sequences contain all of the MC29-specific sequences and 5' helper-related sequences as well as part of the envelope region. The size of the cloned EcoRI fragment is the same as that of the major band in EcoRI-cleaved Q5 DNA that hybridizes to viral sequences. Transfection of the cloned DNA into NIH 3T3 cells revealed that the MC29-specific sequences are functional in that they induce foci of transformed cells with high efficiency.

Mammalian cells contain multiple copies of endogenous type C retroviral DNA sequences. Among these sequences are complete, potentially infectious proviruses, proviral DNA that is expressed only in the form of viral antigens, retroviral segments that may contribute portions of envelope (env) genes during the generation of recombinant polytropic viruses, and many subgenomic viral DNA segments that may not be expressed at all. We have previously reported the identification and molecular cloning of type C retroviral sequences from human DNA and have shown that the partial nucleotide and deduced amino acid sequences of one of the clones obtained (lambda 51) are homologous to Moloney MuLV (MoMuLV) in the gag and pol regions. The lambda 51 clone as well as several others isolated from a human DNA library contained approximately 4.3 kilobases (kb) of retroviral sequences, were deleted in the env region, and were flanked by tandem repeats unlike the long terminal repeats (LTRs) typically found in proviral DNAs (P.E.S., in preparation). We describe here the characterization of a full-length human retroviral clone (lambda 4-1) containing LTR elements as well as a putative env region. DNA-RNA hybridization experiments reveal that human cells contain species of poly(A)+ RNA that anneal to segments of the full-length retroviral DNA clone.

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.

The genetic structure of the McDonough strain of feline sarcoma virus (SM-FeSV) was deduced by analysis of molecularly cloned, transforming proviral DNA. The 8.2-kilobase pair SM-FeSV provirus is longer than those of other feline sarcoma viruses and contains a transforming gene (v-fms) flanked by sequences derived from feline leukemia virus. The order of genes with respect to viral RNA is 5'-gag-fms-env-3', in which the entire feline leukemia virus env gene and an almost complete gag sequence are represented. Transfection of NIH/3T3 cells with cloned SM-FeSV proviral DNA induced foci of morphologically transformed cells which expressed SM-FeSV gene products and contained rescuable sarcoma viral genomes. Cells transformed by viral infection or after transfection with cloned proviral DNA expressed the polyprotein (P170gag-fms) characteristic of the SM-FeSV strain. Two proteolytic cleavage products (P120fms and pp55gag) were also found in immunoprecipitates from metabolically labeled, transformed cells. An additional polypeptide, detected at comparatively low levels in SM-FeSV transformants, was indistinguishable in size and antigenicity from the envelope precursor (gPr85env) of feline leukemia virus. The complexity of the v-fms gene (3.1 +/- 0.3 kilobase pairs) is approximately twofold greater than the viral oncogene sequences (v-fes) of Snyder-Theilen and Gardner-Arnstein FeSV. By heteroduplex, restriction enzyme, and nucleic acid hybridization analyses, v-fms and v-fes sequences showed no detectable homology to one another. Radiolabeled DNA fragments representing portions of the two viral oncogenes hybridized to different EcoRI and HindIII fragments of normal cat cellular DNA. Cellular sequences related to v-fms (designated c-fms) were much more complex than c-fes and were distributed segmentally over more than 40 kilobase pairs in cat DNA. Comparative structural studies of the molecularly cloned proviruses of Synder-Theilen, Gardner-Arnstein, and SM-FeSV showed that a region of the feline-leukemia virus genome derived from the pol-env junction is represented adjacent to v-onc sequences in each FeSV strain and may have provided sequences preferred for recombination with cellular genes.

Naturally elaborated membrane bleb material is frequently observed in cultures of Neisseria gonorrhoeae. This material was purified and analyzed for protein, lipopolysaccharide, and nucleic acid content. The electrophoretic protein profiles of two bleb-rich fractions, called BI and BII, were distinct, with only BII containing lipopolysaccharide and outer membrane proteins I and III. Both fractions contained RNA, circular DNA, and linear DNA. Exogenous pancreatic DNase I appeared to hydrolyze all bleb-associated DNA in fraction BI and the linear DNA in fraction BII. The circular DNA molecules associated with fraction BII resisted digestion. Electron microscopy of the bleb fractions verified their DNA content. Fixing blebs with glutaraldehyde before mounting them for microscopy prevented release of internal DNA. Such fixation produced little change in the micrographs of BI; however, only traces of DNA were observed in fixed BII preparations. Incubation of wild-type gonococci in mixtures of DNase and blebs purified from antibiotic-resistant strains resulted in efficient exchange of penicillinase-specifying R plasmids. Recipients incorporated plasmids independently of endogenous and exogenous chromosomal streptomycin resistance markers. These in vitro results suggest that bleb formation by N. gonorrhoeae may serve to transfer plasmids intercellularly in vivo, perhaps constituting a previously unexplored genetic exchange mechanism in these bacteria.

Enteral adenovirus (EAd) isolates which characteristically fail to propagate in several human cell lines grow readily in 293 cells, an adenovirus type 5 transformed line. EAd infection of 293 cells is associated with high levels of 31S DNA and late viral protein synthesis, the appearance of a progressive adenovirus-like cytopathic effect, and the efficient assembly of progeny virions. The DNA of an EAd strain grown in 293 cells possesses restriction endonuclease profiles similar to those reported for EADs recovered directly from stools. In their ability to replicate efficiently in 293 cells but not in other human cell lines. EAds appear to behave like adenovirus host range mutants.

The two polyoma DNA fragments generated by cleavage with BamHI and EcoRI were cloned in pBR322, and their oncogenic potential was tested in vivo and in vitro. Only recombinant plasmid DNA containing a polyoma DNA fragment which extends clockwise from 58 to 0 map units and include approximately the 5'-proximal half of the early gene region produced tumors in newborn hamsters and transformed rat embryo cells in tissue culture. Southern blotting analysis indicated that the entire 2.2-kilobase polyoma BamHI-EcoRI fragment was intact in both a tumor cell line and a cell line transformed in culture which we examined. The presence of polyoma middle and small T antigen in these lines was demonstrated by immunoprecipitation and tryptic peptide mapping. DNA from a recombinant plasmid containing a polyoma genome deleted between 90 and 4 map units failed to induce tumors or transform cells.

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.

Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo, and transforms fibroblasts and hematopoietic target cells in vitro, We have used recombinant DNA technology to isolate and characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by Southern blotting of restriction endonuclease digests and by electron microscopic visualization of R-loops formed between the cloned DNA and MC29 or helper virus RNA. It was found that the 9.2 kb cloned DNA insert contains approximately 4 kb of viral sequences and 5.2 kb of quail cellular sequences. The viral sequences contain all of the MC29 specific sequences and 5' helper related sequences as well as part of the envelope region. The size of the cloned EcoRI fragment is the same as that of the major band in EcoRI-cleaved Q5 DNA that hybridizes to viral sequences. Transfection of the cloned DNA into NIH 3T3 cells revealed that the MC29-specific sequences are functional in that they induce foci of transformed cells with high efficiency.

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.