Plasmid modifications in a tick‐borne pathogen, <i>Borrelia burgdorferi</i>, cocultured with tick cells

Munderloh, Ulrike G.; Park, Y. ‐J; Dioh, J. M.; Fallon, Ann M.; Kurtti, Timothy J.

doi:10.1111/j.1365-2583.1993.tb00092.x

Cited by 14 publications

(14 citation statements)

References 34 publications

(30 reference statements)

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“…The present study confirms that B. burgdorferi cells multiply when they are cocultured with tick cell lines in L15BS medium but are unable to grow in this medium in the absence of tick cells (25). It has been claimed previously that B. burgdorferi is found both outside and within tick and mammalian host cells (6,12,18,19,22,26).…”

Section: Discussionsupporting

confidence: 86%

Modulation of Borrelia burgdorferi Stringent Response and Gene Expression during Extracellular Growth with Tick Cells

Bugrysheva¹,

Dobrikova²,

Godfrey

et al. 2002

Infect Immun

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Borrelia burgdorferi N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support B. burgdorferi N40 growth. Growth of B. burgdorferi N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in relA/spoT mRNA, an eightfold net decrease in bmpD mRNA, and a fourfold increase in rpsL-bmpD mRNA compared to growth of B. burgdorferi in BSK-H medium. As a result, the polycistronic rpsL-bmpD mRNA level increased from 3 to 100% of the total bmpD message. These observations demonstrate that there are reciprocal interactions between B. burgdorferi and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in B. burgdorferi growing in BSK-H medium is ameliorated in B. burgdorferi growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of B. burgdorferi with tick cells in vitro when it is coupled with genetic methods to isolate and complement B. burgdorferi mutants.Borrelia burgdorferi, the cause of Lyme disease, modulates its gene expression in the presence of host cells. As an example of this modulation, the relative expression of the ospA and ospC genes is altered as B. burgdorferi passes from a tick to a mammalian host (32); a similar down-modulation-up-modulation of OspA and OspC is observed during coculture of B. burgdorferi with tick cells in vitro (27). This oscillation in gene expression may be mediated by a regulatory loop involving changes in the expression of 54 and S , suggesting that B. burgdorferi growing in the presence of tick cells may undergo a series of as-yet-uncharacterized global regulatory changes (36). It also suggests that study of the reciprocal interactions between B. burgdorferi and tick cells could illuminate aspects of gene modulation in B. burgdorferi and the genes and gene products involved (17,36).Flexible adaptation by bacterial pathogens to rapidly changing host environments requires the interplay of regulators generated in response to signals triggered by environmental stimuli. One such adaptive response is the stringent response, a global response triggered by nutritional stress when protein synthesis is blocked by amino acid starvation (10, 13, 34). The stringent response causes up-regulation of protein degradation and amino acid synthesis, down-regulation of nucleic acid and protein synthesis, and alteration of many cellular functions, including transcription, translation, replication, transport, and metabolism, and it is accompanied by slow growth (10, 34). In Escherichia coli, the stringent response involves RelA, a synthase, and SpoT, a hydrolase, which synthesize and degrade, respectively, ppGpp and pppGpp [(p)ppGpp], the alarmon (master regulator, second message) that is the proximate mediator of this response (9, 10). In some bacterial pathogens, such as Mycobacterium t...

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Section: Discussionsupporting

confidence: 86%

Modulation of Borrelia burgdorferi Stringent Response and Gene Expression during Extracellular Growth with Tick Cells

Bugrysheva¹,

Dobrikova²,

Godfrey

et al. 2002

Infect Immun

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“…However, our present and previous analyses of R. felis isolate LSU have revealed the presence of only one plasmid species (7,40). We have shown plasmid loss in serially passaged R. peacockii and previously used the same method to demonstrate plasmid loss and rearrangements in Borrelia burgdorferi strain JMNT, which was serially passaged with cultured tick cells following its isolation from a hamster (29). The Borrelia plasmid changes were accompanied by changes in infectivity, consistent with similar observations made by others (45).…”

Section: Discussionmentioning

confidence: 57%

Plasmids of the pRM/pRF Family Occur in DiverseRickettsiaSpecies

Baldridge

Burkhardt

Felsheim

et al. 2008

Appl Environ Microbiol

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The recent discoveries of the pRF and pRM plasmids of Rickettsia felis and R. monacensis have contravened the long-held dogma that plasmids are not present in the bacterial genus Rickettsia (Rickettsiales; Rickettsiaceae). We report the existence of plasmids in R. helvetica, R. peacockii, R. amblyommii, and R. massiliae isolates from ixodid ticks and in an R. hoogstraalii isolate from an argasid tick. R. peacockii and four isolates of R. amblyommii from widely separated geographic locations contained plasmids that comigrated with pRM during pulsed-field gel electrophoresis and larger plasmids with mobilities similar to that of pRF. The R. peacockii plasmids were lost during long-term serial passage in cultured cells. R. montanensis did not contain a plasmid. Southern blots showed that sequences similar to those of a DnaA-like replication initiator protein, a small heat shock protein 2, and the Sca12 cell surface antigen genes on pRM and pRF were present on all of the plasmids except for that of R. massiliae, which lacked the heat shock gene and was the smallest of the plasmids. The R. hoogstraalii plasmid was most similar to pRM and contained apparent homologs of proline/betaine transporter and SpoT stringent response genes on pRM and pRF that were absent from the other plasmids. The R. hoogstraalii, R. helvetica, and R. amblyommii plasmids contained homologs of a pRM-carried gene similar to a Nitrobacter sp. helicase RecD/TraA gene, but none of the plasmids hybridized with a probe derived from a pRM-encoded gene similar to a Burkholderia sp. transposon resolvase gene.The genus Rickettsia (Rickettsiales; Rickettsiaceae) consists of small gram-negative obligately intracellular alphaproteobacteria associated with eukaryotic hosts. They occur in a wide range of arthropods (38, 39) but are best known as vertebrate pathogens transmitted by blood-feeding arthropods. Rickettsia prowazekii and Rickettsia typhi, transmitted by lice and fleas, respectively, are the etiologic agents of epidemic and endemic typhus. Various species associated with ticks are the agents of spotted fevers occurring around the world, but others found in ticks have not been associated conclusively with human disease (5, 37). Rickettsia species are the closest known microbial relatives of mitochondria (3, 58) and have undergone the reductive genome evolution characteristic of strict endosymbiotic bacteria (2,4,12). Rickettsial genomes are typically 1.1 ϫ 10 6 to 1.5 ϫ 10 6 base pairs in length, with a GC content of approximately 30%, and have high levels of both synteny and noncoding content (12). Many biosynthetic pathways that are present in free-living bacteria have been replaced by transport systems in rickettsiae, which renders them dependent on eukaryotic host cells for essential metabolites (42).The recent discoveries of pRF in Rickettsia felis by wholegenome sequencing (35) and of pRM in Rickettsia monacensis (7) contravened the long-held dogma that plasmids are not present in rickettsiae. The pRM plasmid was identified by pulsed-field gel...

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“…Several isolates carrying 92-to 105-kb plasmids, 40 to 50 kb larger than those typically observed (2-4, 8, 16, 22, 31-35, 39), were identified (Table 1). Only B. burgdorferi R100 had been previously shown to carry a plasmid in this size range (26). Southern blot analyses with an ospA probe (ospA-5Ј-GGCTGCTAACATTT-TGCTTACATGC) revealed that the ospAB operon was carried by the large plasmids in IKA2, HO14 (Fig.…”

mentioning

confidence: 91%

Analysis of linear plasmid dimers in Borrelia burgdorferi sensu lato isolates: implications concerning the potential mechanism of linear plasmid replication

et al. 1996

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The Borrelia genome is composed of a linear chromosome and a number of variable circular and linear plasmids. Atypically large linear plasmids of 92 to 105 kb have been identified in several Borrelia burgdorferi sensu lato isolates and characterized. These plasmids carry the p27 and ospAB genes, which in other isolates reside on a 50-kb plasmid. Here we demonstrate that these plasmids are dimers of the 50-kb ospAB plasmid (pAB50). The 94-kb plasmid from isolate VS116, pVS94, was an exception and did not hybridize with any plasmid gene probes. When this plasmid was used as a probe, homologous sequences in other isolates were not detected, suggesting that it is unique to isolate VS116. These analyses provide insight into the mechanism of linear plasmid replication and the mechanisms by which plasmid variability can arise.Lyme disease, a tick-borne zoonosis (5,7,36,37), is caused by Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii (1,6,19,20,38). Related species of uncertain pathogenic potential, i.e., Borrelia japonica (18) and Borrelia andersonii (21), have also been identified. These five species are collectively referred to as B. burgdorferi sensu lato (BBsl). The BBsl genome is composed of variable linear and circular plasmids (15, 30) and a linear chromosome (11). The ospAB operon is carried on the largest of the linear plasmids, which ranges from 48 to 60 kb (31). We refer to this variably sized plasmid as the 50-kb ospAB plasmid or pAB50. The importance of the BBsl plasmids is underscored by the fact that they are present in all isolates and that they carry genes encoding metabolic enzymes (24). In view of this, the extent of plasmid variability among isolates is surprising. The significance of and the mechanisms involved in generating plasmid variability are not completely understood. Plasmid loss (32), recombination (17,22,28), and lateral transfer of plasmids among BBsl species (22) have been demonstrated to be contributing processes. Here we demonstrate that linear plasmid dimer formation also contributes to plasmid variability and discuss how these findings may provide information concerning the mechanisms of linear plasmid replication.Bacterial isolates analyzed in this study were cultivated at 34ЊC in BSK-H (Sigma) supplemented with 6% rabbit serum. Pulsed-field gel electrophoresis (PFGE) was performed to analyze plasmid content as previously described (21). Several isolates carrying 92-to 105-kb plasmids, 40 to 50 kb larger than those typically observed (2-4, 8, 16, 22, 31-35, 39), were identified (Table 1). Only B. burgdorferi R100 had been previously shown to carry a plasmid in this size range (26). Southern blot analyses with an ospA probe (ospA-5Ј-GGCTGCTAACATTT-TGCTTACATGC) revealed that the ospAB operon was carried by the large plasmids in IKA2, HO14 (Fig. 1), BO23, and R100 and by a 50-kb plasmid in VS116 (data not shown). In IKA2, a second plasmid of 47 kb also bound the probe (Fig. 1); however, after continued in vitro cultivation, this plasmid was lost and an ospAB-hybridizing ...

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Plasmid modifications in a tick‐borne pathogen, Borrelia burgdorferi, cocultured with tick cells

Cited by 14 publications

References 34 publications

Modulation of Borrelia burgdorferi Stringent Response and Gene Expression during Extracellular Growth with Tick Cells

Modulation of Borrelia burgdorferi Stringent Response and Gene Expression during Extracellular Growth with Tick Cells

Plasmids of the pRM/pRF Family Occur in DiverseRickettsiaSpecies

Analysis of linear plasmid dimers in Borrelia burgdorferi sensu lato isolates: implications concerning the potential mechanism of linear plasmid replication

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