Genome-Wide Transposon Mutagenesis of Borrelia burgdorferi for Identification of Phenotypic Mutants

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the leading vector-borne illness in the United States. Many of the genetic factors affecting spirochete morphology and physiology are unknown due to the limited genetic tools available and the large number of open reading frames with unknown functions. By adapting a mariner transposon to function in B. burgdorferi, we have developed a random mutagenesis system that tags the mutated locus for rapid identification. Transposition occurs at saturating levels in B. burgdorferi and appears to be random, targeting both linear and circular replicons. By combining the transposon system with a screen for factors affecting growth rate, mutations were readily identified in genes putatively involved in cell division and chemotaxis and a hypothetical open reading frame involved in outer membrane integrity. The successful adaptation of a mariner transposon to function in B. burgdorferi should aid in identifying virulence factors and novel gene products related to spirochete physiology.     

Genetic and physiological characterization of 23S rRNA and ftsJ mutants of Borrelia burgdorferi isolated by mariner transposition

Borrelia burgdorferi contains one 16S rRNA gene and two tandem sets of 23S and 5S rRNA genes located in a single chromosomal region. This unusual rRNA gene organization has been speculated to be involved in the slow growth of this organism. Because we were repeatedly unable to isolate a 23S ribosomal mutant in B. burgdorferi by allelic exchange, we developed a transposition mutagenesis system for this bacterium. To this end, Himar1 transposase is expressed in B. burgdorferi from a resident plasmid containing an erythromycin resistance marker, and this strain is then electroporated with suicide plasmids containing mariner transposons and kanamycin resistance genes expressible in B. burgdorferi. This system permitted us to generate hundreds of erythromycin/kanamycin-resistant B. burgdorferi clones with each of three suicide plasmids. DNA sequencing of several kanamycin-resistant clones generated with one of the suicide plasmids showed stable and random insertion of the transposon into the B. burgdorferi chromosomal and plasmid genome. One mutant was inactivated in rrlA (23S rRNA), another in ftsJ (rrmJ). rrlA disruption had no effect on growth rate under a wide range of culture conditions, but disruption of ftsJ interfered significantly with growth rate and bacterial morphology. These data show it is possible to isolate random and stable B. burgdorferi transposition mutants for physiological analysis of this pathogenic spirochete.     

Random insertional mutagenesis of Leptospira interrogans, the agent of leptospirosis, using a mariner transposon

The recent availability of the complete genome sequences of Leptospira interrogans, the agent of leptospirosis, has allowed the identification of several putative virulence factors. However, to our knowledge, attempts to carry out gene transfer in pathogenic Leptospira spp. have failed so far. In this study, we show that the Himar1 mariner transposon permits random mutagenesis in the pathogen L. interrogans. We have identified genes that have been interrupted by Himar1 insertion in 35 L. interrogans mutants. This approach of transposon mutagenesis will be useful for understanding the spirochetal physiology and the pathogenic mechanisms of Leptospira, which remain largely unknown.     

aadA confers streptomycin resistance in Borrelia burgdorferi

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.     

The plasmids of Borrelia burgdorferi: essential genetic elements of a pathogen

The spirochete Borrelia burgdorferi, the causative agent of Lyme disease, has an unusual genome comprised of a linear chromosome and the largest plasmid complement of any characterized bacterium. Certain plasmid-encoded elements are required for virulence and viability, both in vitro and in vivo. The genetic tools to manipulate B. burgdorferi are sufficiently developed for precise molecular genetic investigations. B. burgdorferi now represents a prime system with which to address basic questions of plasmid biology and plasmid contributions to bacterial virulence and disease pathogenesis.     

Development of a Transposon Mutagenesis System in the Oral Spirochete Treponema denticola

Here, we report successful transposon mutagenesis in the oral spirochete Treponema denticola. A modified Himar1 transposon, including a new antibiotic selection cassette for T. denticola, generated mutations affecting cell division, transport, and chemotaxis, among other processes. This random mutagenesis system should facilitate research on the biology and pathogenesis of this spirochete, which is associated with human periodontal diseases.     

Heterologous transposition in <Emphasis Type="Italic"> Ustilago maydis</Emphasis>

The phytopathogenic basidiomycete Ustilago maydis has become a model system for the analysis of plant-pathogen interactions. The genome sequence of this organism will soon be available, increasing the need for techniques to analyse gene function on a broad basis. We describe a heterologous transposition system for U. maydis that is based on the Caenorhabditis transposon Tc1, which is known to function independently of host factors and to be active in evolutionarily distant species. We have established a nitrate reductase based two-component counterselection system to screen for Tc1 transposition. The element was shown to be functional and transposed to several different locations in the genome of U. maydis. The insertion pattern observed was consistent with the proposed general mechanism of Tc1/mariner integration and constitutes a proof of principle for the first heterologous transposition system in a basidiomycete species. By mapping the insertion site context to known genomic sequences, Tc1 insertion events were shown to occur on different chromosomes, but exhibit a preference for non-coding regions. Only 20% of the insertions were found in putative open reading frames. The establishment of this system will permit efficient gene tagging in U. maydis and possibly also in other fungi.     

The generation of enzymatically active plasmin on the surface of spirochetes

The spirochete Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted to the host by a feeding Ixodid tick. The spirochete subsequently disseminates through the skin, enters the bloodstream, and becomes systemic. A potential mechanism for this invasiveness was identified with the discovery that B. burgdorferi can bind components of the plasminogen activation system (PAS). The methodology for analyzing the generation of enzymatically active plasmin on the surface of this organism is given, and applied to measure spirochete viability, strain differences, and breakdown of extracellular matrix (ECM) macromolecules. Plasmin acquisition by B. burgdorferi was measured photometrically by a specific chromogenic substrate. The growth of B. burgdorferi in culture was not affected by the presence of active plasmin on the spirochete surface. Plasmin-coated B. burgdorferi degraded the purified (ECM) components fibronectin, laminin, and vitronectin, but not collagen. The addition of B. burgdorferi with surface plasmin to a radiolabeled, native ECM resulted in degradation of noncollagenous protein, as measured by release of solubilized radioactivity. Breakdown of purified ECM components or native ECM did not occur after exposure to untreated spirochetes or spirochetes treated with uPA or PLG alone. These results provide in vitro evidence that enzymatically active plasmin on the surface of B. burgdorferi may be partially responsible for its invasiveness.     

Isolation and characterization of FecA- and FeoB-mediated iron acquisition systems of the spirochete Leptospira biflexa by random insertional mutagenesis

The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.     

Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi

Borrelia burgdorferi contains abundant circular and linear plasmids, but the mechanism of replication of these extrachromosomal elements is unknown. A B. burgdorferi 9 kb circular plasmid (cp9) was amplified in its entirety by the polymerase chain reaction and used to construct a shuttle vector that replicates in Escherichia coli and B. burgdorferi. A 3.3 kb region of cp9 containing three open reading frames was used to construct a smaller shuttle vector, designated pBSV2. This vector was stably maintained in B. burgdorferi, indicating that all elements necessary for autonomous replication are probably located on this 3.3 kb fragment. A non-infectious B. burgdorferi strain was efficiently transformed by pBSV2. Additionally, infectious B. burgdorferi was also successfully transformed by pBSV2, indicating that infectious strains of this important human pathogen can now be genetically manipulated.     

The many faces of Borrelia burgdorferi

In this review we describe several genetic regulatory mechanisms adopted by the agent of Lyme disease, Borrelia burgdorferi, to sense and adapt to different host and environmental conditions either in vitro or in vivo. This regulation results in the increased or decreased synthesis of several proteins whose levels are believed to play key roles in the ability of B. burgdorferi to cycle between both arthropod and mammalian hosts. Moreover, the differential synthesis of these proteins serves to modulate the response of B. burgdorferito signals in the requisite host and may also, in some cases, function as virulence determinants of this spirochete. Elucidation of these mechanisms will help in the understanding of the pathogenicity of B. burgdorferi as well as aid in identifying proteins that are important during different stages of infection.     

The highly modified microcin peptide plantazolicin is associated with nematicidal activity of Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 has been shown to stimulate plant growth and to suppress the growth of plant pathogenic organisms including nematodes. However, the mechanism underlying its effect against nematodes remains unknown. In this study, we screened a random mutant library of B. amyloliquefaciens FZB42 generated by the mariner transposon TnYLB-1 and identified a mutant strain F5 with attenuated nematicidal activity. Reversible polymerase chain reaction revealed that three candidate genes RAMB_007470, yhdY, and prkA that were disrupted by the transposon in strain F5 potentially contributed to its decreased nematicidal activity. Bioassay of mutants impaired in the three candidate genes demonstrated that directed deletion of gene RBAM_007470 resulted in loss of nematicidal activity comparable with that of the F5 triple mutant. RBAM_007470 has been reported as being involved in biosynthesis of plantazolicin, a thiazole/oxazole-modified microcin with hitherto unknown function. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) analyses of surface extracts revealed that plantazolicin bearing a molecular weight of 1,354 Da was present in wild-type B. amyloliquefaciens FZB42, but absent in the ΔRABM_007470 mutant. Furthermore, bioassay of the organic extract containing plantazolicin also showed a moderate nematicidal activity. We conclude that a novel gene RBAM_007470 and its related metabolite are involved in the antagonistic effect exerted by B. amyloliquefaciens FZB42 against nematodes.     

Repetition, conservation, and variation: the multiple cp32 plasmids of Borrelia species

Members of the spirochete genus Borrelia contain large numbers of extrachromosomal DNAs. Sequence analysis of the B. burgdorferi strain B31 genome indicated that its many plasmids contain large quantities of repeated sequences, the most obvious of which are the cp32 plasmid family. Individual spirochetes may carry nine or more different, but homologous, cp32 plasmids. Every other species of Borrelia examined thus far also contains multiple plasmids related to the B. burgdorferi cp32s. These plasmids are arguably the best characterized of all the borrelial plasmids, and epitomize the apparent redundancy evident in the many plasmids carried by these bacteria. Despite their extensive similarities, cp32 plasmids contain some open reading frames whose sequences often vary between plasmids, and which encode proteins synthesized by the bacteria during vertebrate infection. In this review, we analyze the hypervariable and conserved regions of the cp32 plasmid family, and discuss possible reasons why borreliae harbor multiple gene paralogs.     

Hidden in plain sight: Borrelia burgdorferi and the extracellular matrix

Borrelia burgdorferi, the tick-transmitted etiologic agent of Lyme borreliosis, can colonize and persist in multiple tissue sites despite vigorous host immune responses. The extracellular matrix appears to provide a protective niche for the spirochete. Recent studies in mice suggest that B. burgdorferi interacts in various ways with collagen and its associated molecules, exploiting molecular and structural features to establish microcolonial refugia. Better knowledge of the genetic and structural bases for these interactions of B. burgdorferi with the extracellular matrix will be required before an understanding of the persistence of B. burgdorferi in the tissues and development of chronic infections can be achieved.     

Genetic variation at the vlsE locus of Borrelia burgdorferi within ticks and mice over the course of a single transmission cycle

The Lyme disease spirochete, Borrelia burgdorferi, causes a persistent infection in the vertebrate host even though infected animals mount an active immune response against the spirochete. One strategy used by the spirochete to evade vertebrate host immunity is to vary the structure and expression of outer membrane antigens. The vlsE locus represents the best-studied example of antigenic variation in B. burgdorferi. During vertebrate host infection, recombination between the active vlsE locus and silent, partial vlsE copies leads to gene conversion events and the generation of novel alleles at the expression site. In the present study, we followed a population of B. burgdorferi organisms moving through vertebrate host and tick stages to complete one transmission cycle. The major goal of the study was to determine if the vlsE locus was subject to different selective pressure and/or recombination frequency at different stages of the spirochete's life cycle. We report here that the vlsE genetic diversity generated within the rodent host was maintained through the larval and nymphal tick stages. Therefore, naturally infected ticks are likely to transmit spirochete populations with multiple vlsE alleles into naive vertebrate hosts. Although vlsE genetic diversity in mice was maintained through tick stages, the dominant vlsE alleles were different between tick stages as well as between individual ticks. We propose that population-level bottlenecks experienced by spirochetes, especially during the larval-to-nymphal molt, are responsible for individual infected ticks harboring different dominant vlsE alleles. Although vlsE genetic diversity is maintained through tick stages, the VlsE protein is unlikely to be of functional importance in the vector, because the protein was expressed by very few (<1%) bacteria in the vector.     

Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks

Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.     

转座子挽救法对转座子突变菌株中插入位点的定位分析

为寻找谷氨酸棒杆菌转座子插入突变菌株中的转座子插入位点,采用了转座子挽救法对转座子及其插入位点附近的序列进行分离,并测定插入位点相邻DNA序列,获得了三个转座子插入位点DNA序列,其中一个是柠檬酸合成酶基因,另两个为目前未知基因,暂命名为orfA和orfB。该方法简便易行,是分析转座子插入位点的理想方法。