Efficient targeted mutagenesis in Borrelia burgdorferi

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB(r), a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A(1). The utility of the coumermycin-resistant gyrB(r) gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.     

Genetic transformation of the Lyme disease agent Borrelia burgdorferi with coumarin-resistant gyrB.

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.     

gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi.

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.     

New antibiotic resistance cassettes suitable for genetic studies in Borrelia burgdorferi

In this report we describe two distinct approaches to develop new antibiotic resistance cassettes that allow for efficient selection of Borrelia burgdorferi transformants. The first approach utilizes fusions of borrelial flagellar promoters to antibiotic resistance markers from other bacteria. The AACC1 gene, which encodes a gentamicin acetyltransferase, conferred a high level of gentamicin resistance in B. Burfdorferi when expressed from these promoters. No cross-resistance occurred between this cassette and the kanamycin resistance cassette, which was previously developed in an analogous fashion. A second and different approach was taken to develop an efficient selectable marker that confers resistance to the antibiotic coumermycin A1. A synthetic gene was designed from the GYRB301 allele of the coumermycin-resistant B. Burgdorferi strain B31-NGR by altering the coding sequence at the wobble position. The resulting gene, GYRB(SYN), encodes a protein identical to the product of GYRB301, but the genes share only 66% nucleotide identity. The nucleotide sequence of GYRB(SYN)is sufficiently divergent from the endogenous B. Burgdorferi GYRB gene to prevent recombination between them. The cassettes described in this paper improve our repertoire of genetic tools in B. Burgdorferi. These studies also provide insight into parameters governing recombination and gene expression in B. Burgdorferi.     

Homologous Recombination as the Main Mechanism for DNA Integration and Cause of Rearrangements in the Filamentous Ascomycete Ashbya Gossypii

A slow and a fast growth phenotype were observed after transformation of the phytopathogenic fungus Ashbya gossypii using a plasmid carrying homologous DNA and as selectable marker the Tn903 aminoglycoside resistance gene expressed from a strong A. gossypii promoter. Transformations with circular plasmids yielded slowly and irregularly growing geneticin-resistant mycelia in which 1% of nuclei contained plasmid sequences. Occasionally, fast growing sectors appeared which were shown to be initiated by homologous integration of the transforming DNA. Transformants obtained with plasmids linearized within the homology region immediately exhibited fast radial growth. In all 28 transformants analyzed plasmid DNA was integrated homologously. Such apparent lack of nonhomologous recombination has so far not been observed in filamentous ascomycetes. In 14 transformants two to four tandemly integrated plasmid copies were found. They underwent several types of genetic changes, mainly in the older mycelium: excision of whole plasmid copies and rearrangements within the integrated DNA (inversions and deletions). These internal rearrangements involved 360-bp inverted repeats, remnants of IS-elements flanking the resistance gene, and 156-bp direct repeats, originating from the strong A. gossypii promoter. Improved vectors lacking sequence repetitions were constructed and used for stable one-step gene replacement in A. gossypii.     

Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element.

We have cloned three copies of a repeated DNA segment from Borrelia burgdorferi sensu stricto strain B31, present on both circular and linear plasmids of this and other B. burgdorferi sensu lato strains. The DNA sequences are characterized by a highly homologous segment containing two open reading frames (ORFs), ORF-A and ORF-B. Five additional ORFs can be found on the slightly less homologous flanking sequences: ORF-G on the opposite strand upstream of ORF-A, and ORF-C, ORF-D, ORF-E, and ORF-F downstream of ORF-B. The 4.6-kb-long element containing ORF-A through ORF-E is flanked by approximately 180-bp-long imperfect inverted repeats (IRs). The putative gene product of ORF-C displays homology to proteins involved in plasmid maintenance in a number of gram-positive and gram-negative bacteria. ORF-E features several short, highly homologous direct repeats. ORF-A, ORF-B, and ORF-D are homologous to three ORFs on a recently described 8.3-kb circular plasmid of Borrelia afzelii Ip21 that are flanked by similar IRs (J. J. Dunn, S. R. Buchstein, L.-L. Butler, S. Fisenne, D. S. Polin, B. N. Lade, and B. J. Luft, J. Bacteriol. 176:2706-2717,1994). ORF-C and ORF-E, however, are missing from this region on the Ip21 plasmid. Furthermore, the repeated DNA element as defined by the IRs is present in opposite orientations relative to the flanking sequences on the B31 and Ip21 plasmids.     

Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation.

We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome. A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12. Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker. Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector.     

Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.     

Characterization of a suppressor mutation complementing an acid-sensitive mutation in Streptococcus mutans

We isolated a spontaneous suppressor mutant complementing the acid-sensitive phenotype of Streptococcus mutans strain Tn-1, a mutant previously generated in this laboratory, defective in the activity of the dgk-encoded putative undecaprenol kinase. A relatively simple genetic method was developed to identify the suppressor mutation, based on selection for transformants containing two closely linked markers: a selectable allele of the unknown suppressor gene and an antibiotic resistance gene introduced on a suicide plasmid at random sites into the chromosome via homologous recombination. While we have not actually identified the original suppressor mutation, another mutated gene restoring acid resistance has been isolated, which suggests a possible mechanism of suppression.     

Genetic diversity and the absence of regional differences of Borrelia garinii as demonstrated by ospA and ospB gene sequence analysis

Unfed adult Ixodes persulcatus ticks were collected from four locations of Nagano and Hokkaido in Japan. Infected Borrelia garinii were investigated by PCR-RFLP of the ospA and ospB gene sequences. The primer set amplified an approximately 1.6-kb DNA fragment (0.7-kb in some strains), and BsrI, BstYI, or NlaIII digestion of the product resulted in six distinctively different PCR-RFLP groups and two independent borrelial strains. The representatives in each PCR-RFLP group and individuals from the borrelial strains were sequenced, and their deduced amino acid sequences were aligned. A neighbor-joining phylogenetic analysis showed that the B. garinii OspA or OspB sequences were each divided into three major clusters including isolates from both the Nagano and Hokkaido locations. There was no local difference in OspA/B sequences between Nagano and Hokkaido. The osp gene of Borrelia burgdorferi sensu lato is highly heterogeneous, and this was also confirmed by our sequence analysis. Some strains of the different PCR-RFLP groups had closely related OspA sequences, while the OspB sequences of these strains were quite different. These findings suggested intraspecies gene exchange and recombination events between the two genes in B. garinii.     

Stable transformation of the cyanobacterium Synechocystis sp. PCC 6803 induced by UV irradiation.

Irradiation of the photoheterotrophic cyanobacterium Synechocystis sp. PCC 6803 with low levels of UV light allows for stable, integrative transformation of these cells by heterologous DNA. In this system, transformation does not rely on an autonomously replicating plasmid and is independent of homologous recombination. Cells treated with UV light in the absence of DNA and cells given DNA but not exposed to UV do not yield antibiotic-resistant colonies in platings of up to 2 X 10(8) cells. Optimal conditions for this UV-induced transformation are described. Analysis of the transformants indicates that (i) only a segment of the introduced plasmid is found in the DNA of the transformed cells; (ii) in independently isolated clones, DNA insertion apparently occurs at different sites in the chromosome; and (iii) hybridization data suggest that insertion in one of the transformants may have occurred into a region of the chromosome that is repeated or that integration of plasmid DNA may have been accompanied by a rearrangement or duplication of DNA sequences near the insertion site. DNA isolated from the primary transformants as well as a cloned fragment containing the UV-inserted plasmid sequence and flanking cyanobacterial DNA transform wild-type cells at a high frequency (5.0 X 10(-4) and 1.5 X 10(-5), respectively). Possible mechanisms of this transformation system are discussed, as are the potential uses of this system as an integrative cloning-complementation vector and as a mutagenic agent in which the genetic lesion is already tagged with a selectable marker.     

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.     

New plasmid vectors for specific gene targeting in Spiroplasma citri

In Spiroplasma citri gene inactivation through homologous recombination has been achieved by using the replicative, oriC plasmid pBOT1 as the disruption vector. However, plasmid recombination required extensive passaging of the transformants and, in most cases, recombination occurred at oriC rather than at the target gene. In the current study, we describe a new vector, in which the oriC fragment was reduced to the minimal sequences able to promote plasmid replication. Using this vector to inactivate the motility gene scm1 showed that size reduction of the oriC fragment did increase the frequency of recombination at the target gene. Furthermore, to avoid extensive passaging of the transformants, we developed a strategy in which the selective, tetracycline resistance phenotype can only be expressed once the plasmid has integrated into the chromosome by one single crossover recombination at the target gene. As an example, targeting of the spiralin gene is described.     

Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

'Integron'-bearing vectors: a method suitable for stable chromosomal integration in highly restrictive corynebacteria.

A pBR322-derived plasmid (pCGL107) that carries the Corynebacterium melassecola ATCC17965 analogue of Escherichia coli gdhA gene (encoding glutamate dehydrogenase), was introduced into the related strain, Brevibacterium lactofermentum CGL2002, by electroporation and integrated into its chromosome by homologous recombination. However, pCGL107 cannot integrate into C. melassecola, since the host restriction prevents successful electroporation by E. coli-modified DNA. Nevertheless, B. lactofermentum-modified replicative plasmid DNA can be transformed by electroporation into C. melassecola; thus pCGL519-2, a shuttle plasmid that carries the C. melassecola analogue of E. coli gltA (encoding citrate synthase), was extracted from the former host and electroporated into the latter. Rare restriction sites conveniently placed in pCGL519-2 were used to recover a replicon-less cartridge called 'integron', that contains a selectable marker and gtlA within a single fragment. Integron prepared from pCGL519-2 DNA which had been extracted from C. melassecola, and thus, was capable of eluding the C. melassecola restriction barrier(s), was successfully electroporated into this host. The molecular analysis of the resulting transformants suggests that they result from the integration of a single circular integron molecule by homologous recombination between the gltA regions of the host genome and the integron. These transformants were stable for 30 generations in the absence of selection.     

High frequency vector-mediated transformation and gene replacement in Tetrahymena.

Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene knockout in Tetrahymena. As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co-express a gene encoding a cycloheximide resistant ribosomal protein.