Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903phikan, carries a cryptic kan gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903phikan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903phikan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903phikan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903phikan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903phikan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903phikan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.     

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity.     

Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative.

A transposon mutagenesis procedure functional in the gram-negative swine pathogen Actinobacillus pleuropneumoniae was developed for the first time. The technique involved the use of a suicide conjugative plasmid, pLOF/Km, carrying a mini-Tn10 with an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible transposase located outside the mobile element (M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172:6557-6567, 1990). The plasmid was mobilized from Escherichia coli to A. pleuropneumoniae through the RP4-mediated broad-host-range conjugal transfer functions provided by the chromosome of the donor strain. When IPTG was present in the mating medium, A. pleuropneumoniae CM5 transposon mutants were obtained at a frequency of 10(-5), while no mutants were detected in the absence of IPTG. Since the frequency of conjugal transfer of the RP4 plasmid from E. coli to A. pleuropneumoniae CM5 was found to be as low as 10(-4), the above result indicated that the expression level of the transposase was a critical factor for obtaining a workable efficiency of transposon mutagenesis. The transposon insertions occurred at random, as determined by Southern blotting of chromosomal DNA of randomly selected mutants and by the ability to generate mutants defective for the selected phenotypes. Almost all the mutants analyzed resulted from a single insertion of the Tn10 element. About 1.2% of the mutants resulted from the cointegration of pLOF/Km into the A. pleuropneumoniae chromosome. The applicability of this transposon mutagenesis system was verified on other A. pleuropneumoniae strains of different serotypes. The usefulness of this transposon mutagenesis system in genetic studies of A. pleuropneumoniae is discussed.     

Identification of differentially regulated francisella tularensis genes by use of a newly developed Tn5-based transposon delivery system

Francisella tularensis is the etiologic agent of an intracellular systemic infection of the lymphatic system in humans called tularemia. The organism has become the subject of considerable research interest due to its classification as a category A select agent by the CDC. To aid genetic analysis of this pathogen, we have constructed a temperature-sensitive Tn5-based transposon delivery system that is capable of generating chromosomal reporter fusions with lacZ or luxCDABE, enabling us to monitor gene expression. Transposition is catalyzed by the hyperactive Tn5 transposase, whose expression is driven by the Francisella groES promoter. When high-temperature selection (42 degrees C) is applied to a bacterial culture carrying the transposon delivery plasmid, approximately 0.1% of the population is recovered with Tn5 insertions in the chromosome. Nucleotide sequence analysis of a sample of mutants revealed that the insertions occur randomly throughout the chromosome. The kanamycin-selectable marker of the transposon is also flanked by FLP recombination target sequences that allow deletion of the antibiotic resistance gene when desired. This system has been used to generate transposon mutant libraries for the F. tularensis live vaccine strain as well as two different virulent F. tularensis strains. Chromosomal reporters delivered with the transposon were used to identify genes upregulated by growth in Chamberlain's defined medium. Genes in the fsl operon, reported to be involved in iron acquisition, as well as genes in the igl gene cluster were among those identified by the screen. Further experiments implicate the ferric uptake regulator (Fur) protein in the negative regulation of fsl but not igl reporters, which occurs in an iron-dependent manner. Our results indicate that we have created a valuable new transposon that can be used to identify and characterize virulence genes in F. tularensis strains.     

Idaten is a new cold-inducible transposon of Volvox carteri that can be used for tagging developmentally important genes

A cold-inducible transposon called Jordan has previously been used to tag and recover genes controlling key aspects of Volvox development, including the process called inversion. In a search for additional genes, we isolated 17 new inversionless mutants from cultures grown at 24 degrees (the temperature that activates Jordan transposition). These mutants were stable at 32 degrees, but generated revertants at 24 degrees . DNA blots revealed that one mutant had a transposon unrelated to Jordan inserted in invA ("inversionless A"). This new transposon, which we named Idaten, has terminal inverted repeats (TIRs) beginning with CCCTA, and upon insertion it creates a 3-bp target-site duplication. It appears to belong to the CACTA superfamily of class II DNA transposons, which includes En/Spm. No significant open reading frames were in the Idaten sequence, but we retrieved another element with Idaten-type TIRs encoding a protein similar to the En/Spm transposase as a candidate for an Idaten-specific transposase. We found that in five of the new inversionless strains we could not find any Jordan insertions causing the phenotype to possess insertions of an Idaten family member in a single locus (invC). This clearly indicates that Idaten is a potentially powerful alternative to Jordan for tagging developmentally important genes in Volvox.     

Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature-tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild-type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O-antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.     

Transposon-tagging identifies novel pathogenicity genes in Fusarium graminearum

With the increase of sequenced fungal genomes, high-throughput methods for functional analyses of genes are needed. We assessed the potential of a new transposon mutagenesis tool deploying a Fusarium oxysporum miniature inverted-repeat transposable element mimp1, mobilized by the transposase of impala, a Tc1-like transposon, to obtain knock-out mutants in Fusarium graminearum. We localized 91 mimp1 insertions which showed good distribution over the entire genome. The main exception was a major hotspot on chromosome 2 where independent insertions occurred at exactly the same nucleotide position. Furthermore insertions in promoter regions were over-represented. Screening 331 mutants for sexual development, radial growth and pathogenicity on wheat resulted in 19 mutants (5.7%) with altered phenotypes. Complementation with the original gene restored the wild-type phenotype in two selected mutants demonstrating the high tagging efficiency. This is the first report of a MITE transposon tagging system as an efficient mutagenesis tool in F. graminearum.     

Novel genomic locus with atypical G+C content that is required for extracellular polysaccharide production and virulence in Xanthomonas oryzae pv. oryzae.

Three exopolysaccharide (EPS)- and virulence-deficient mutants of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, were isolated by Tn5 mutagenesis. These insertions are not located within the gum gene cluster. A 40-kb cosmid clone that restored EPS production and virulence to all three mutants was isolated, and the three transposon insertions were localized to contiguous 4.3- and 3.5-kb EcoRI fragments that are included in this clone. Sequence data indicate that two of the transposon insertions are in genes that encode a putative sugar nucleotide epimerase and a putative glycosyl transferase, respectively; the third insertion is located between the glycosyl transferase gene and a novel open reading frame (ORF). A 5.5-kb genomic region in which these three ORFs are located has a G+C content of 5-1.7%, quite different from the G+C content of approximately 65.0% that is typical of X. oryzae pv. oryzae. Homologues of this locus have not yet been reported in any other xanthomonad.     

Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli.

We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon insertions in essential genes.     

Counterselection and Co-Delivery of Transposon and Transposase Functions for <Emphasis Type="Italic">Sleeping Beauty</Emphasis>-Mediated Transposition in Cultured Mammalian Cells

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.     

Identification of Streptococcus agalactiae virulence genes in the neonatal rat sepsis model using signature-tagged mutagenesis

Group B streptococcal (GBS) infections are the most common cause of bacterial sepsis in the immediate newborn period. Apart from the capsule, the factors required for survival of GBS in the host are not well defined. In this study, signature-tagged transposon mutagenesis (STM) was used to identify genes required for growth and survival of GBS in a neonatal rat sepsis infection model. Approximately 1600 transposon mutants were screened in pools of 80 mutants, and approximately 120 mutants defective for survival in the animal host were identified. We successfully cloned and sequenced DNA flanking the transposon insertions from 92 of the mutants. Fifty per cent of the mutants had transposon insertions in genes with homologues in the public databases, whereas the remaining 50% had transposon insertions in genes with unknown function. A significant proportion of the avirulent mutants had transposon insertions in genes encoding transport-associated or regulatory proteins or in genes involved in cell surface metabolism, emphasizing the significance of these functions for in vivo survival of GBS. Overall, STM analysis revealed GBS genomic loci that encode a wide variety of functional gene classes, underscoring the diversity of bacterial processes required for the infection process. Currently, the function of the genes identified during the screening can only be inferred by homology to previously described genes. However, a number of the genes identified in this study have been shown to correlate with virulence in other pathogens. A virulence of a subset of mutants identified during the screening was confirmed by performing competitive index assays and lethal dose assays. This represents the first report of a genome-wide scan for virulence factors in GBS. The identified genes will further our understanding of the pathogenesis of GBS infections and may represent targets for intervention or lead to the development of novel therapies.     

In vivo random mutagenesis of Bacillus subtilis by use of TnYLB-1, a mariner-based transposon

This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either sigmaA- or sigmaB-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kan r cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Erm r gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10(-2)) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a "TA" dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo- clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.     

Tipping the balance between replicative and simple transposition

The bacterial insertion sequence IS903 has the unusual ability to transpose both replicatively and non-replicatively. The majority of products are simple insertions, while co-integrates, the product of replicative transposition, occur at a low frequency (<0.1% of simple insertions). In order to define the critical steps that determine the outcome of IS903 transposition, we have isolated mutants that specifically increase the rate of replicative transposition. Here we show that the nucleotide immediately flanking the transposon influences both overall transposition frequency and co-integrate formation. In particular, when the 3'-flanking nucleotide is A, co-integrates are increased 500-fold compared with a 3' C. In addition, we have isolated five transposase mutants that increase replicative transposition. These residues are close to the catalytic residues and are thus likely to be part of the active site. These are the first transposase mutations described that affect the product of transposition. Our results are consistent with the hypothesis that a delay in cleavage of the 5'-flanking DNA will increase the effective half-life of the 3'-nicked transposon intermediate and consequently enhance co-integrate formation.     

Genetic analysis of the marine manganese-oxidizing Bacillus sp. strain SG-1: protoplast transformation, Tn917 mutagenesis, and identification of chromosomal loci involved in manganese oxidation.

Mature spores of the marine Bacillus sp. strain SG-1 bind and oxidize manganese(II), thereby becoming encrusted with a manganese(IV) oxide. Both the function and mechanism of this oxidation are unknown, although evidence suggests that spore coat proteins are involved. To further study this phenomenon, methods of genetic analysis were developed for SG-1. By a modified protoplast transformation procedure, SG-1 was transformed (approximately 100 transformants per micrograms of DNA) with several different plasmids of gram-positive origin. Transposon Tn917, delivered on the temperature-sensitive plasmid pLTV1, was used to generate mutants of SG-1. Conditions were established that allowed 98% plasmid loss and insertions to be recovered at a frequency of 10(-3). Each mutant was found to be the result of a single insertion event. Restriction analysis of 27 mutants that do not oxidize manganese but still sporulate localized 17 of the insertions within two regions of the chromosome (termed Mnx regions), and a physical map of these regions was generated. Analysis of 18 transposon integrants in which manganese oxidation was unaffected revealed random transposon integration, with none of their insertions mapping within the Mnx regions. The Mnx regions were cloned from wild-type SG-1, and the largest region, carried on the lactococcal plasmid pGK13, was used to complement in trans one of the nonoxidizing mutants. These results demonstrate that the Mnx regions encode factors that are required for the oxidation of manganese, and this represents the first report identifying genes involved in bacterial manganese oxidation.     

Genetic footprinting in bacteria

In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage lambda transposon delivery system.     

Helicobacter pylori mutagenesis by mariner in vitro transposition

We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ-marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments.     

Site-selected insertion of the transposon Tc1 into a Caenorhabditis elegans myosin light chain gene.

We used the polymerase chain reaction to detect insertions of the transposon Tc1 into mlc-2, one of two Caenorhabditis elegans regulatory myosin light chain genes. Our goals were to develop a general method to identify mutations in any sequenced gene and to establish the phenotype of mlc-2 loss-of-function mutants. The sensitivity of the polymerase chain reaction allowed us to identify nematode populations containing rare Tc1 insertions into mcl-2. mlc-2::Tc1 mutants were subsequently isolated from these populations by a sib selection procedure. We isolated three mutants with Tc1 insertions within the mlc-2 third exon and a fourth strain with Tc1 inserted in nearby noncoding DNA. To demonstrate the generality of our procedure, we isolated two additional mutants with Tc1 insertions within hlh-1, the C. elegans MyoD homolog. All of these mutants are essentially wild type when homozygous. Despite the fact that certain of these mutants have Tc1 inserted within exons of the target gene, these mutations may not be true null alleles. All three of the mlc-2 mutants contain mlc-2 mRNA in which all or part of Tc1 is spliced from the pre-mRNA, leaving small in-frame insertions or deletions in the mature message. There is a remarkable plasticity in the sites used to splice Tc1 from these mlc-2 pre-mRNAs; certain splice sites used in the mutants are very different from typical eukaryotic splice sites.     

The Hermes transposon of Musca domestica and its use as a mutagen of Schizosaccharomyces pombe

Transposon mutagenesis allows for the discovery and characterization of genes by creating mutations that can be easily mapped and sequenced. Moreover, this method allows for a relatively unbiased approach to isolating genes of interest. Recently, a system of transposon based mutagenesis for Schizosaccharomyces pombe became available. This mutagenesis relies on Hermes, a DNA transposon from the house fly that readily integrates into the chromosomes of S. pombe. The Hermes system is distinct from the retrotransposons of S. pombe because it efficiently integrates into open reading frames. To mutagenize S. pombe, cells are transformed with a plasmid that contains a drug resistance marker flanked by the terminal inverted repeats of Hermes. The Hermes transposase expressed from a second plasmid excises the resistance marker with the inverted repeats and inserts this DNA into chromosomal sites. After S. pombe with these two plasmids grow 25 generations, approximately 2% of the cells contain insertions. Of the cells with insertions, 68% contain single integration events. The protocols listed here provide the detailed information necessary to mutagenize a strain of interest, screen for specific phenotypes, and sequence the positions of insertion.