Homology of variable major protein genes between Borrelia hermsii and Borrelia miyamotoi

Abstract Antigenic variation has been studied in detail for the etiological agent of relapsing fever, Borrelia hermsii . The variable major proteins (vmps) are found at its cell surface, enabling it to avoid the host's immune response. We have cloned and sequenced the vmp -gene ( vmp )-like sequences from the Borrelia miyamotoi strains HT31 and FR64b and the deduced amino acid sequences were compared with the published vmp proteins vmp3, vmp24, and vmp33 of B. hermsii . The sequences were aligned and revealed pairwise sequence identities ranging from 45 to 51%, and differences were scattered throughout the sequences. Southern hybridization using the cloned vmp -like sequence of strain HT31 as a probe suggested that the vmp homologues reside on the linear plasmids of B. miyamotoi . The probe hybridized weakly with B. hermsii linear plasmids and restriction digests. These results suggest that B. miyamotoi has sequences resembling the vmp genes in B. hermsii .     

Subtelomeric expression regions of Borrelia hermsii linear plasmids are highly polymorphic

Borrelia hermsii, a relapsing fever agent, undergoes multiphasic antigenic variation to evade its host's immune response. Serotype specificity is determined by variable membrane lipoproteins, Vmps, which are expressed from genes located near the end of a linear plasmid. Using the polymerase chain reaction and primers representing the promoter of the active vmp and a conserved telomeric sequence, we characterized the subtelomeric expression regions of the 25 known serotypes of strain HS1. The distance from the promoter to the telomere fell into three size classes of approximately 1.0, 1.5, and 2.5 kilobases. In the sequenced serotypes the size differences were accounted for by variable lengths of the vmp genes and intervening sequences between 3' end of the vmp gene and the start of a downstream homology block. The degree of nucleotide identity between different vmp genes, or between the different 3' flanking DNA varied from 39-78%. Thus, there is length and sequence variability not only between vmp genes themselves but also between the 3' flanking regions of vmp genes.     

The variable antigens Vmp7 and Vmp21 of the relapsing fever bacterium Borrelia hermsii are structurally analogous to the VSG proteins of the African trypanosome

The relapsing fever agent Borrelia hermsii avoids the host's immune response by the strategy of multiphasic antigenic variation. A given Borrelia cell can express one of a number of alleles for polymorphic outer-membrane proteins, known as Vmp proteins. The genes for the variant-specific Vmp proteins of serotypes 7 and 21 of B. hermsii strain HS1 were sequenced. The genes, which were designated vmp7 and vmp21, were obtained from populations of borreliae before and after a switch in serotypes from 7 to 21. The analysis showed that vmp7 and vmp21 are 77% identical in terms of their coding sequence. The deduced translation products of vmp7 and vmp21 are polypeptides of 369 (37.2 kD) and 364 amino acids (37.1 kD), respectively. Vmp7 and Vmp21 have sequence features of prokaryotic lipoproteins and are processed as such during expression in E. coli. The secondary structure predictions of the Vmp proteins reveals analogous structures to the VSG proteins of the African trypanosome.     

Variable antigen genes of the relapsing fever agent Borrelia hermsii are activated by promoter addition

Borrelia hermsii, an agent of relapsing fever, avoids the host's immune response by means of multiphasic antigenic variation. Serotype specificity is determined by variable antigens called the Vmp lipoproteins. Through recombination between linear plasmids a formerly silent vmp gene replaces another vmp gene at a telomeric expression locus. We examined strain HS1 borreliae before and after a switch from serotype 7 to serotype 21. The nucleotide sequences of 5' regions of silent and expressed vmp7 and vmp21 were determined. Silent and active vmp7 and vmp21 genes shared a block of homologous sequences surrounding their 5' ends. Sequences upstream of silent vmp7 and vmp21 genes lacked the promoter and substantially differed from each other. In this antigenic switch a vmp gene was activated by a recombination that placed it downstream of a promoter.     

Size conversion of a linear plasmid in the relapsing fever agent Borrelia duttonii

Borreliae have genomes composed of both linear and circular replicons. We have characterized the organization of linear DNA molecules from the Borrelia duttonii strain Ly. It contains a linear one megabase chromosome and 12 linear plasmids of 11 to 200 kb in size. A variant of the strain obtained after successive in vitro cultivation in BSKII medium had a 69 kb molecule instead of the 44 kb linear plasmid. No detectable differences in the growth rates and cellular structures were found. Southern hybridization using the vsp33 gene sequence from Borrelia hermsii as a probe showed that both plasmids (69 and 44 kb molecules) contained a similar part of the sequence. The spirochetes of the parental strain cause erythrocytes to aggregate in mice blood, but the variant did not form such aggregates and seemed to have lost its infectivity in mice. Size conversion of the linear plasmid may be associated with the host-parasite relationship in mammals.     

Linear plasmids of Borrelia burgdorferi have a telomeric structure and sequence similar to those of a eukaryotic virus.

Spirochetes of the genus Borrelia have double-stranded linear plasmids with covalently closed ends. The physical nature of the terminal connections was determined for the 16-kb linear plasmid of the B31 strain of the Lyme disease agent Borrelia burgdorferi. Native telomeric fragments representing the left and right ends of this plasmid were isolated and subjected to Maxam-Gilbert sequence analysis. At the plasmid ends the two DNA strands formed an uninterrupted, perfectly palindromic, AT-rich sequence. This Borrelia linear plasmid consisted of a continuous polynucleotide chain that is fully base paired except for short single-stranded hairpin loops at each end. The left and right telomeres of the 16-kb plasmid were identical for 16 of the first 19 nucleotide positions and constituted an inverted terminal repeat with respect to each other. The left telomere of the 49-kb plasmid of strain B31 was identical to the corresponding telomere of the 16-kb plasmid. Different-sized plasmids of other strains of B. burgdorferi also contained sequences homologous to the left end of the 16-kb plasmid. When the borrelia telomeres were compared with telomeric sequences of other linear double-stranded DNA replicons, sequence similarities were noted with poxviruses and particularly with the iridovirus agent of African swine fever. The latter virus and a Borrelia sp. share the same tick vector. These findings suggest that the novel linear plasmids of Borrelia originated through a horizontal genetic transfer across kingdoms.     

Intragenic recombination and a chimeric outer membrane protein in the relapsing fever agent Borrelia hermsii.

The spirochete Borrelia hermsii, a relapsing fever agent, evades the host's immune response through multiphasic antigenic variation. Antigen switching results from sequential expression of genes for serotype-specific outer membrane proteins known as variable major proteins (Vmp's); of the 25 serotypes that have been identified for the HS1 strain, serotypes 7 and 21 have been studied in greatest detail. In the present study, an atypical variant was predominant in the relapse from a serotype 21 infection in mice; relapse cells were bound by monoclonal antibodies specific for Vmp21 as well as antibodies specific for Vmp7. In Western blots (immunoblots), the variant had a single Vmp that was reactive with monoclonal antibodies representing both serotypes. The gene encoding this Vmp, vmp7/21, was cloned and characterized by restriction mapping and sequence analysis to determine the likely recombination event. Whereas the 5' end of vmp7/21 was identical to that of vmp21, its 3' end and flanking sequences were identical to the 3' end of vmp7. Unlike other vmp genes examined thus far, the vmp7/21 gene existed only in an expressed form; a silent, storage form of the gene was not detected. We conclude that the vmp7/21 gene was created by an intragenic recombination between the formerly expressed vmp21 gene and a silent vmp7 gene. This finding suggests that the lack of cross-reactivity between variants, which is usually observed, results from immunoselection against variants possessing chimeric Vmp's rather than from a switching mechanism that excludes partial gene replacements.     

Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii.

Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two species, behaved as linear duplex DNA molecules under different electrophoretic conditions. A variant of strain HSI of B. hermsii had a 180-kb circular instead of linear plasmid. There were no detectable differences in the growth rates or in the expression of cellular proteins between cells bearing linear forms and those bearing circular forms of the plasmid. The conversion to a circular conformation of monomeric length was demonstrated by the introduction of strand breaks with irradiation, restriction endonuclease analysis, and direct observation of the DNA molecules by fluorescent microscopy. Consideration of different models for the replication of linear DNA suggests that circular intermediates may be involved in the replication of linear replicons in Borrelia spp.     

Linear- and circular-plasmid copy numbers in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.     

Plasmid location of Borrelia purine biosynthesis gene homologs.

The Lyme disease spirochete Borrelia burgdorferi must survive in both its tick vector and its mammalian host to be maintained in nature. We have identified the B. burgdorferi guaA gene encoding GMP synthetase, an enzyme involved in de novo purine biosynthesis that is important for the survival of bacteria in mammalian blood. This gene encodes a functional product that will complement an Escherichia coli GMP synthetase mutant. The gene is located on a 26-kb circular plasmid, adjacent to and divergent from the gene encoding the outer surface protein C (OspC). The guaB gene homolog encoding IMP dehydrogenase, another enzyme in the purine biosynthetic pathway, is adjacent to guaA. In Borrelia hermsii, a tick-borne relapsing fever spirochete, the guaA and guaB genes are located on a linear plasmid. These are the first genes encoding proteins of known function to be mapped to a borrelial plasmid and the only example of genes encoding enzymes involved in the de novo purine biosynthesis pathway to be mapped to a plasmid in any organism. The unique plasmid location of these and perhaps other housekeeping genes may be a consequence of the segmented genomes in borreliae and reflect the need to adapt to both the arthropod and mammalian environments.     

Large Linear Plasmids of Borrelia Species That Cause Relapsing Fever

Borrelia species of relapsing fever (RF) and Lyme disease (LD) lineages have linear chromosomes and both linear and circular plasmids. Unique to RF species, and little characterized to date, are large linear plasmids of ∼160 kb, or ∼10% of the genome. By a combination of Sanger and next-generation methods, we determined the sequences of large linear plasmids of two New World species: Borrelia hermsii, to completion of its 174-kb length, and B. turicatae, partially to 114 kb of its 150 kb. These sequences were then compared to corresponding sequences of the Old World species B. duttonii and B. recurrentis and to plasmid sequences of LD Borrelia species. The large plasmids were largely colinear, except for their left ends, about 27 kb of which was inverted in New World species. Approximately 60% of the B. hermsii lp174 plasmid sequence was repetitive for 6 types of sequence, and half of its open reading frames encoded hypothetical proteins not discernibly similar to proteins in the database. The central ∼25 kb of all 4 linear plasmids was syntenic for orthologous genes for plasmid maintenance or partitioning in Borrelia species. Of all the sequenced linear and circular plasmids in Borrelia species, the large plasmid''s putative partition/replication genes were most similar to those of the 54-kb linear plasmids of LD species. Further evidence for shared ancestry was the observation that two of the hypothetical proteins were predicted to be structurally similar to the LD species'' CspA proteins, which are encoded on the 54-kb plasmids.     

Function and evolution of plasmid-borne genes for pyrimidine biosynthesis in Borrelia spp

The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.     

nifV is contiguous to nifHDK in Frankia strain FaC1

The organization of genes with the capacity to code for four proteins involved in nitrogen fixation in Frankia strain FaC1 was determined by restriction fragment mapping and nucleotide sequence analysis. Analysis of the 44-kb genomic cosmid clone pFAH 1. isolated from a cosmid library made from Frankia strain FaCl, resulted in the identification of a 7.2-kb Pst I fragment to which Klebsiella nif H, nif D and nif K probes hybridized. This nif -hybridizing fragment was subcloned and analyzed by restriction fragment mapping. Further subcloning of the 7.2-kb fragment and subsequent sequence analysis of approximately 6.8 kb revealed the presence of six open reading frames (ORFs). Four of these ORFs have the potential to code for nif V-, nif H-, nif D- and nif K-like gene products and the two others are unidentified ORFs. The organization of the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes, but the positioning of the nif V-like gene relative to the nif HDK cluster differs, A consensus nif -promoter-like sequence, found 5'to nif H. was not detected upstream of the nif V-like gene. Nine copies of a 7-bp direct repeat were found 5'to ORFA.     

Extensive interplasmidic duplications change the virulence phenotype of the relapsing fever agent Borrelia turicatae

The relapsing fever agent Borrelia turicatae has two antigenically distinct serotypes, A and B, which differ in their variable small proteins (Vsps) and in their degree of virulence and neurotropism in mice. Each Vsp gene (vspA or vspB) had an expression-linked copy that was unique to the serotype expressing it. This was located on one linear plasmid, which was defined by the upstream sequence. The archived copies of vspA and vspB were each located on different linear plasmids that were the same in both serotypes. In this feature, the mechanism of antigenic variation is similar to that of another relapsing fever agent, B. hermsii. However, in other features, the mechanisms of the two organisms differ. The expressed and archived loci for vspA and vspB of B. turicatae were near the centre of linear plasmids instead of near the telomeres. The vspA and vspB expression loci were duplicate copies of their respective silent loci: from the vsp itself to at least 13-14 kb downstream. Despite the extensive interplasmidic duplications and the internal position of the expression locus, the only detectable difference between serotypes A and B was in whether they expressed VspA or VspB.     

The mitochondrial genome of the Basidiomycete fungus Pleurotus ostreatus (oyster mushroom)

In this study, the full mitochondrial genome of a basidiomycete fungus, Pleurotus ostreatus, was sequenced and analyzed. It is a circular DNA molecule of 73 242 bp and contains 44 known genes encoding 18 proteins and 26 RNA genes. The protein-coding genes include 14 common mitochondrial genes, one ribosomal small subunit protein 3 gene, one RNA polymerase gene and two DNA polymerase genes. In addition, one RNA and one DNA polymerase genes were identified in a mitochondrial plasmid. These two genes show relatively low similarities to their homologs in the mitochondrial genome but they are nearly identical to the known mitochondrial plasmid genes from another Pleurotus ostreatus strain. This suggests that the plasmid may mediate the horizontal gene transfer of the DNA and RNA polymerase genes into mitochondrial genome, and such a transfer may be an ancient event. Phylogenetic analysis based on the cox1 ORFs verified the traditional classification of Pleurotus ostreatus among fungi. However, the discordances were observed in the phylogenetic trees based on the six cox1 intronic ORFs of Pleurotus ostreatus and their homologs in other species, suggesting that these intronic ORFs are foreign DNA sequences obtained through HGT. In summary, this analysis provides valuable information towards the understanding of the evolution of fungal mtDNA.