An abundant chloroplast protein is implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants. The product of the Escherichia coli groEL gene is essential for cell viability and is required for the assembly of bacteriophage capsids. Sequencing of the groEL gene and the complementary cDNA encoding the chloroplast protein has revealed that these proteins are evolutionary homologues which we term 'chaperonins'. Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes. Assisted post-translational assembly of oligomeric protein structures is emerging as a general cellular phenomenon.

Environmentally responsive synthesis of surface proteins represents a hallmark of the infectious cycle of the Lyme disease agent, Borrelia burgdorferi. Here we created and analyzed a B. burgdorferi mutant lacking outer-surface protein C (OspC), an abundant Osp that spirochetes normally synthesize in the tick vector during the blood meal and down-regulate after transmission to the mammal. We demonstrate that B. burgdorferi strictly requires OspC to infect mice but not to localize or migrate appropriately in the tick. The induction of a spirochetal virulence factor preceding the time and host in which it is required demonstrates a developmental sequence for transmission of this arthropod-borne pathogen.

A major obstacle to studying the functions of particular gene products in the mouse-tick infectious cycle of Borrelia burgdorferi has been an inability to knock out genes in pathogenic strains. Here, we investigated conditions for site-directed mutagenesis in B31 MI, the low-passage-number, infectious B. burgdorferi strain whose genome was sequenced. We inactivated several plasmid and chromosomal genes in B31 MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31 MI based on colony phenotype, growth rate, plasmid content, protein profile, and transformability. Significantly, several B31 MI clones that were not subjected to mutagenesis but that lacked particular plasmids also exhibited defects at various stages in the infectious cycle. Therefore, the high degree of clonal polymorphism within B31 MI complicates the assessment of the contributions of individual genes to the observed phenotypes of the mutants. Our results indicate that B31 MI is not an appropriate strain background for genetic studies in infectious B. burgdorferi, and a well-defined isogenic clone is a prerequisite for targeted mutagenesis. To this end, we derived several wild-type clones from B31 MI that were infectious for mice, and gene inactivation was successful in one of these clones. Due to the instability of the genome with in vitro propagation, careful monitoring of plasmid content of derived mutants and complementation of inactivated genes will be crucial components of genetic studies with this pathogen.Lyme disease is caused by Borrelia burgdorferi, a spirochete transmitted by ticks of the genus Ixodes and maintained within an enzootic cycle between the tick vector and mammalian hosts, most importantly small rodents (7,11,19). The clinical manifestations of this zoonosis can include a multisystem disorder affecting skin and joints and the nervous, lymphoreticular, and cardiovascular systems (39,40).The organization of the B. burgdorferi genome is unique among bacteria in that the genome is composed of a linear chromosome and a large number of linear and circular plasmids (8, 14). The complete genome sequence of an infectious B. burgdorferi isolate, the type strain B31, identified 21 linear and circular plasmids (8). In vitro propagation of B. burgdorferi can lead to plasmid loss and concurrent loss of infectivity for mice (3,21,22,30,33). Although increasing evidence suggests that certain Borrelia plasmids are important for infection in mice (18,25), this hypothesis has not been experimentally verified, and the roles of most plasmid-encoded genes in the infectious cycle are unknown. A number of plasmid-and chromosomally encoded genes have been inactivated in the highpassage-number, noninfectious clone B31-A (4,5,12,17,20,43,45,46), but gene inactivation in a low-passage-number, infectious strain background has not been reported.Here, we investigate conditions for site-directed mutagenesis in B31 MI, the low-passage-number, inf...

This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.

We have characterized seven different 32-kb circular plasmids carried by Borrelia burgdorferi isolate B31. Restriction endonuclease recognition site mapping and partial sequencing of these plasmids indicated that all seven are probably closely related to each other throughout their lengths and have substantial relationships to cp8.3, an 8.3-kb circular plasmid of B. burgdorferi sensu lato isolate Ip21. With the addition of the seven 32-kb plasmids, this bacterial strain is known to carry at least 10 linear and 9 circular plasmids. Variant cultures of B. burgdorferi B31 lacking one or more of the 32-kb circular plasmids are viable and, at least in some cases, infectious. We have examined a number of different natural isolates of Lyme disease borreliae and found that all of the B. burgdorferi sensu stricto isolates and most of the B. burgdorferi sensu lato isolates tested appear to carry multiple 32-kb circular plasmids related to those of B. burgdorferi B31. The ubiquity of these plasmids suggests that they may be important in the natural life cycle of these organisms. They may be highly conjugative plasmids or prophage genomes, which could prove to be useful in genetically manipulating B. burgdorferi.Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic ailment of humans that is spread through the bite of certain species of Ixodes ticks (13, 61). As a spirochete, B. burgdorferi is a member of a morphologically and phylogenetically distinct order of eubacteria (11,28,(42)(43)(44)70). Although classically defined as gram-negative organisms (28), recent phylogenetic studies based on rRNA sequences indicated that the spirochetes are as distantly related to the gramnegative Escherichia coli as they are to gram-positive bacteria (42,70).One of the most striking differences between B. burgdorferi and other bacteria is its unusual genome, which includes a small (approximately 1 Mb) linear chromosome (12,16,19,21,23,41) and numerous linear and circular plasmids, sometimes approaching 20 different plasmids in one bacterium (7,8,10,22,27,30,35,57,65,71). A curious feature of these different plasmids is that they often appear to share regions of homologous DNA (58,60,65,71,73,74). Homologs of DNA sequences mapped to circular plasmids have even been found on linear plasmids (74), although in a related bacterium, Borrelia hermsii, at least one plasmid exists in both linear and circular forms (24), blurring the distinction between these two forms of DNA. Several of the plasmids that these bacteria harbor appear to be present in all natural isolates; therefore, the term minichromosome may be a more apt description of their nature. For example, a 49-to 54-kb linear plasmid and a 26-kb circular plasmid, which carry the outer surface protein genes ospAB and ospC, respectively, are ubiquitous (7,34,37,52,54,63,71). It is not known whether members of either of these two plasmid families have similar overall gene orders in different bacterial isolates, but their nearly invariant sizes support the idea that they...

A novel procedure for saturation mutagenesis of cloned DNA was used to obtain more than 100 single base substitutions within the promoter of the mouse beta-major globin gene. The effects of these promoter substitutions on transcription were determined by transfecting the cloned mutant genes into HeLa cells on plasmids containing an SV40 transcription enhancer, and measuring the levels of correctly initiated beta-globin transcripts after 2 days. Mutations in three regions of the promoter resulted in a significant decrease in the level of transcription: (i) the CACCC box, located between -87 and -95, (ii) the CCAAT box, located between -72 and -77, and (iii) the TATA box, located between -26 and -30 relative to the start site of transcription. In contrast, two different mutations in nucleotides immediately upstream from the CCAAT box resulted in a 3- to 3.5-fold increase in transcription. With two minor exceptions, single base substitutions in all other regions of the promoter had no effect on transcription. These results precisely delineate the cis-acting sequences required for accurate and efficient initiation of beta-globin transcription, and they establish a general approach for the fine structure genetic analysis of eukaryotic regulatory sequences.

We have identified four loci in Borrelia burgdorferi B31 that contain open reading frames capable of encoding six proteins that are related to the antigenic proteins OspE and OspF. We have designated these proteins Erp, for OspEF-related protein, and named their respective genes erp. The erpA and erpB genes are linked, as are erpC and erpD, and the pairs probably constitute two operons. The erpG and erpH genes appear to be monocistronic. The ErpA and ErpC proteins are expressed by B. burgdorferi B31 in culture and are recognized by a polyclonal antiserum raised against the OspE protein of B. burgdorferi N40. The four erp loci are each located on different 32-kb circular plasmids that contain additional DNA sequences that are homologous to each other and to an 8.3-kb circular plasmid of B. burgdorferi sensu lato Ip21. All four 32-kb plasmids can be maintained within a single bacterium, which may provide a model for the study of plasmid replication and segregation in B. burgdorferi.Borrelia burgdorferi is a member of the order Spirochaetales, the spirochetes, an order that is phylogenetically and morphologically distinct from such well-characterized bacteria as Escherichia coli and Bacillus subtilis (8,24,34,35,53). B. burgdorferi bacteria naturally contain a wide variety of plasmids (4,13,25,40,42,45,47,54), yet nothing is known about the mechanisms by which plasmids are maintained in these bacteria. Several observations suggest that the small DNA species of B. burgdorferi and other spirochetes may be fundamentally different from the plasmids of other bacteria, the most significant being that there are linear plasmids in B. burgdorferi (4,5,7,13,22,23,42). Also, two B. burgdorferi genes involved in purine biosynthesis are located on a circular plasmid (29), which appears to be inconsistent with the classical definition of a plasmid as a nonessential extrachromosomal element. Other plasmids of B. burgdorferi appear to be capable of undergoing recombination to form multimeric plasmids (25, 31), and there have been reports of DNA sequences located on several different plasmids within the same bacterium (46,47,(54)(55)(56).B. burgdorferi is the causative agent of Lyme disease, an increasingly common ailment of humans and several other mammals (15,48). Infection of a mammal by B. burgdorferi is generally accompanied by the production of antibodies directed against a limited number of bacterial antigens (17,19,41,52), several of which have been identified as surface-exposed lipoproteins. At least three of these lipoproteins, OspC, OspE, and OspF, are differentially expressed by B. burgdorferi, being produced in greater quantities by cultured bacteria that are shifted from 23 to 32ЊC than by those maintained at the lower temperature (43, 49). For OspC, at least, this appears to be related to induction of specific protein synthesis by the spirochete in ticks following feeding upon a warm-blooded animal (43). Temperature-dependent differential expression of bacterial proteins that are involved in host infection has been o...

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