Identification of a hotspot for transformation of Neisseria meningitidis by shuttle mutagenesis using signature-tagged transposons

Shuttle mutagenesis using signature-tagged transposons was employed to generate a library of individually tagged mutants of the Neisseria meningitidis strain B1940, which belongs to serogroup B. The use of tagged transposons allowed us to monitor for enrichment for single mutants during the process of shuttle mutagenesis, by amplification of the tags and subsequent sequence determination. Enrichment of a single clone occurred during the transformation of the meningococci with transposon-containing plasmid DNA. Sequence determination around the site of transposon insertion revealed that the transposon had mutagenized a previously unknown locus, which was designated hrtA (high rate of transformation). hrtA-mediated transformation was independent of TnMax5 and tag sequences, and it most probably involved recombination events. The hrtA locus is restricted to meningococci and gonococci and is present in few apathogenic neisserial species. Chromosomal mapping of hrtA and six further hrt sites revealed a random distribution of highly transforming DNA fragments on the meningococcal chromosome. In conclusion, our data demonstrate that shuttle mutagenesis of naturally competent bacteria using signature-tagged transposons allows the isolation of chromosomal DNA fragments, which exhibit a high transformation efficiency, and which, therefore, are likely to be involved in horizontal gene transfer.     

Construction of a Large Signature-Tagged Mini-Tn5 Transposon Library and Its Application to Mutagenesis of Sinorhizobium meliloti

Sinorhizobium meliloti genome sequence determination has provided the basis for different approaches of functional genomics for this symbiotic nitrogen-fixing alpha-proteobacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. This method is efficient for single genes; however, it is laborious and time-consuming if it is used on a large scale. Here, we used a signature-tagged transposon mutagenesis method that allowed analysis of the survival and competitiveness of many mutants in a single experiment. A novel set of signature tags characterized by similar melting temperatures and G+C contents of the tag sequences was developed. The efficiencies of amplification of all tags were expected to be similar. Thus, no preselection of the tags was necessary to create a library of 412 signature-tagged transposons. To achieve high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, nonredundant sets of signature-tagged S. meliloti mutants for subsequent experiments, 12,000 mutants were constructed, and insertion sites for more than 5,000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns.     

Random shuttle mutagenesis: gonococcal mutants deficient in pilin antigenic variation

Shuttle mutagenesis has been adapted to randomly mutate the genome of Neisseria gonorrhoeae (gono-coccus; Gc). A size-restricted plasmid library of Gc strain FA1090 was mutated with the mini-transposon mTnEGNS. Randomness was tested by checking for transposon insertion bias between vector and insert DNA, Gc transformation efficiency of individual mutated clones, and representation of unique clones before and after Gc transformation with a mutated pool of DNA. Mutants created by random shuttle mutagenesis were screened, using a colony-based polymerase chain reaction assay, for the ability to undergo pilin antigenic variation. Out of 8064 mutants screened, 22 unique transposon insertion mutants were found to be antigenic variation deficient (Avd). The Avd mutants were separated into five types according to recombination defect-associated phenotypes, including colony growth, natural DNA transformation competence, and repair of DNA damage caused by ultraviolet radiation.     

Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti

Sinorhizobium meliloti genome sequence determination has provided the basis for different approaches of functional genomics for this symbiotic nitrogen-fixing alpha-proteobacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. This method is efficient for single genes; however, it is laborious and time-consuming if it is used on a large scale. Here, we used a signature-tagged transposon mutagenesis method that allowed analysis of the survival and competitiveness of many mutants in a single experiment. A novel set of signature tags characterized by similar melting temperatures and G+C contents of the tag sequences was developed. The efficiencies of amplification of all tags were expected to be similar. Thus, no preselection of the tags was necessary to create a library of 412 signature-tagged transposons. To achieve high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, nonredundant sets of signature-tagged S. meliloti mutants for subsequent experiments, 12,000 mutants were constructed, and insertion sites for more than 5,000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns.     

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Summary Fructose was shown to be phosphorylated by a specific phosphoenolpyruvatc-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose Ell. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.     

Tagged Mutagenesis and Gene-trap in the Moss, Physcomitrella patens by Shuttle Mutagenesis

The moss, Physcomitrella patens has been used as a useful materialin many fields, because of its simple body plan, ease of genetargeting, and other reasons. Although many mutants have beenreported, no method to isolate the corresponding genes was reported.We developed a gene tagging and gene-trap system in P. patensby using the shuttle mutagenesis technique, which has been usedin the budding yeast. In 5264 tagged lines, 203 mutants withaltered developmental or morphological phenotypes were obtained.In 129 of 4757 gene-trap lines, ß-glucuronidase (GUS)activity was detected in some tissue. Although multiple copiesof a tag were detected in many tagged lines by Southern analyses,most copies are likely integrated at the same locus accordingto PCR analyses.     

Use of the multipurpose transposon Tn<Emphasis Type="Italic">KPK2</Emphasis> for the mutational analysis of chromosomal regions upstream and downstream of the<Emphasis Type="Italic"> sipF</Emphasis> gene in<Emphasis Type="Italic"> Bradyrhizobium japonicum</Emphasis>

The DNA regions upstream and downstream of the Bradyrhizobium japonicum gene sipF were cloned by in vivo techniques and subsequently sequenced. In order to study the function of the predicted genes, a new transposon for in vitro mutagenesis, TnKPK2, was constructed. This mutagenesis system has a number of advantages over other transposons. TnKPK2 itself has no transposase gene, making transposition events stable. Extremely short inverted repeats minimize the length of the transposable element and facilitate the determination of the nucleotide sequence of the flanking regions. Since the transposable element carries a promoterless phoA reporter gene, the appearance of functional PhoA fusion proteins indicates that TnKPK2 has inserted in a gene encoding a periplasmic or secreted protein. Although such events are extremely rare, because the transposon has to insert in-frame, in the correct orientation, and at an appropriate location in the target molecule, a direct screening procedure on agar indicator plates permits the identification of candidate clones from large numbers of colonies. In this study, TnKPK2 was used for the construction of various symbiotic mutants of B. japonicum. One of the mutant strains, A2-10, which is defective in a gene encoding a protein that comigrates with bacterioferritin (bcpB), was found to induce the formation of small and ineffective nodules.Communicated by A. Kondorosi     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Construction of Signature-tagged Mutant Library in Mesorhizobium loti as a Powerful Tool for Functional Genomics

Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology.Key words: Mesorhizobium loti, signature-tagged mutagenesis, mutant library, reverse genetics     

Analysis of Tn916-induced mutants ofClostridium acetobutylicum altered in solventogenesis and sporulation

Summary The conjugative transposon Tn916 was used for mutagenesis ofClostridium acetobutylicum ATCC 824. Tetracycline-resistant mutants were screened for loss of granulose synthesis and five classes of granulose mutants, that contained single transposon insertions, were identified on the basis of altered solvent production. Class 1 mutants did not make acetone or butanol, lacked activity of enzymes induced during solventogenesis, and did not sporulate, indicating that they are regulatory mutants. The class 2 mutant strains also did not produce acetone but did form small amounts of butanol and ethanol while the class 3 mutants produced low amounts of all solvents. Class 4 and 5 mutants produced essentially the same or higher amounts of solvents than the parent strain. Transposon insertions in the class 1 mutants were used as markers for in vitro synthesis of flanking chromosomal DNA using Tn916-specific primers. The DNA fragments were labeled to produce specific probes. Transposon insertion sites in the chromosomes of 13 different class 1 regulatory mutants were compared by hybridization of the specific probes to Southern blots of restriction endonuclease-digested parental chromosomal DNA. Insertions in two mutants appeared to be, in the same region of the chromosome. These results predict, that multiple regulatory elements are required to induce solvent production and sporulation.     

Use of Ac as an insertional mutagen in Arabidopsis

A pilot-scale transposon mutagenesis experiment using a modified autonomous Activator (Ac) element, AcΔNael, was carried out in Arabidopsis thaliana. Four different transformants carrying Ac elements in different and defined genomic locations were used to generate 1000 plants carrying approximately 500 independent germinal transposition events. These plants were then selfed and the 1000 families screened in tissue culture and soil for phenotypic mutants. Fifty different families segregated mutations in their progeny. Preliminary Southern blot analysis of 29 families which segregated mutant progeny, showed that 28 had a transposed Ac. Six of the families were further tested for linkage between the transposed Ac and the mutant phenotype, and instability of the putatively tagged locus. Two of the mutants were shown to be tagged as they were tightly linked to a transposed Ac, and somatic and germinal reversion was associated with loss of Ac. One other mutant locus was shown to be closely linked to a transposed Ac, and therefore was likely to be tagged. The remaining three mutations were not tagged as they were not linked to a transposed Ac. In two of the tagged mutants Ac had transposed to closely linked sites, while in a third mutant the co-segregating Ac had transposed to a site which was not tightly linked to the donor T-DNA. Multiple insertions into the DIF1 locus were found, due to the preferential transposition of Ac to a linked site.     

Alterations in DNA methylation and genome structure in two rice mutant lines induced by high pressure

By using high-pressure treatment, two mutant lines were obtained from a genetically stable japonica rice cultivar Bijing38. Genomic DNA of the mutant lines, together with the original line (Bijing38), was either undigested or digested by Hpa II/Msp I, and then subjected to molecular analysis using two markers, ISSR and RAPD. Results indicated that changes in the PCR amplification profiles of both markers are apparent in the two mutant lines compared with the original rice cultivar, suggesting that there had been both sequence changes and DNA methylation modifications in the mutant lines. Southern blot analysis using diverse sequences, including two cellular genes (S2 and S3), a set of retrotransposons (Osr7, Osr36, Tos19 and more), and a MITE transposon family (mPing and Pong), confirmed the results, and indicated that changes in DNA methylation pattern, genomic structure, and possible activation of some transposons indeed occurred in the mutant lines. Moreover, these changes are stably maintained through selfed generations and in different organs. Thus, our results indicate that it is possible to obtain stable mutants in rice by high pressure treatments, and the molecular basis of the mutants may include both genetic and epigenetic changes. Therefore, high hydrostatic pressure seems a promising approach for plant mutagenesis.     

Signature-Tagged Transposon Mutagenesis Studies Demonstrate the Dynamic Nature of Cecal Colonization of 2-Week-Old Chickens by Campylobacter jejuni

We have constructed plasmids to be used for in vitro signature-tagged mutagenesis (STM) of Campylobacter jejuni and used these to generate STM libraries in three different strains. Statistical analysis of the transposon insertion sites in the C. jejuni NCTC 11168 chromosome and the plasmids of strain 81-176 indicated that their distribution was not uniform. Visual inspection of the distribution suggested that deviation from uniformity was not due to preferential integration of the transposon into a limited number of hot spots but rather that there was a bias towards insertions around the origin. We screened pools of mutants from the STM libraries for their ability to colonize the ceca of 2-week-old chickens harboring a standardized gut flora. We observed high-frequency random loss of colonization proficient mutants. When cohoused birds were individually inoculated with different tagged mutants, random loss of colonization-proficient mutants was similarly observed, as was extensive bird-to-bird transmission of mutants. This indicates that the nature of campylobacter colonization in chickens is complex and dynamic, and we hypothesize that bottlenecks in the colonization process and between-bird transmission account for these observations.     

Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata

Abstract Molecular genetic studies of halophilic eubacteria have been limited by the lack of a suitable method for mutagenesis. To overcome this, we established a transposon mutagenesis procedure for the ectoine-producing, halophilic bacterium Halomonas elongata . We used suicide plasmids pSUP101 and pSUP102-Gm to introduce the transposons Tn5 and Tn7732 respectively into H. elongata via Escherichia coli SM10 mediated conjugation. Our finding that H. elongata is sensitive to aminoglycoside antibiotics at low salinity enabled us to apply transposons that mediate kanamycin resistance. The insertions of transposon In 1732 occurred at different sites in the chromosome of H. elongata , as proved by Southern hybridization analysis. Phenotypic analysis revealed that different auxotrophic and salt sensitive mutants were generated by mutagenesis with transposon Tn 1732 . To our knowledge this is the first report of a successful application of a transposon for direct generalized mutagenesis in a halophilic eubacterium.     

Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus

Four mutants of Staphylococcus aureus strain Newman that were defective in the fibrinogen receptor (clumping factor) were isolated by transposon Tn917 mutagenesis. Southern hybridization analysis of the mutants identified transposon-host DNA junction fragments, one of which was cloned and used to generate a probe to identify and clone the wild-type clumping factor locus (clfA). The mutants failed to form clumps in soluble fibrinogen and adhered poorly to polymethylmethacrylate (PMMA) coverslips coated with fibrinogen. A single copy of the clfA gene, when introduced into the chromosome of the mutant strains, fuily compiemented the ciumping deficiency of these strains and restored the ability of these mutants to adhere to fibrinogen-coated PMMA. in addition, the cloned clfA gene on a shuttle plasmid aiiowed the weakiy ciumping strain 8325-4 to form clumps with the same avidity as the wild-type strain Newman and also significantly enhanced the adherence of 8325-4 strains. Thus the formation of clumps in soluble fibrinogen correlated with adherence of bacteria to solid-phase fibrinogen. The clfA gene encodes a fibrinogen-binding protein with an apparent molecular mass of c. 130 kDa. The amino acid sequence of the protein was deduced from the DNA sequence; it was predicted that a 896 residue protein (molecular mass 92 kDa) would be expressed. The putative ClfA protein has features that suggest that it is associated with the ceil surface. Furthermore it contains a novel 308 residue region comprising dipeptide repeats predominantly of Asp and Ser ending 28 residues upstream from the LPXTG motif common to wall-associated proteins. Significant homology was found between the ClfA protein and the fibronectin-binding proteins of S. S. aureus, particularly in the N-and C-termini.     

Isolation and Characterisation of Chemotactic Mutants of Chlamydomonas reinhardtii obtained by Insertional Mutagenesis

The swimming behaviour of the green flagellated protist Chlamydomonas reinhardtii is influenced by several different external stimuli including light and chemical attractants. Common components are involved in both the photo- and chemo-sensory transduction pathways, although the nature and organisation of these pathways are poorly understood. To learn more about the mechanism of chemotaxis in Chlamydomonas, we have generated nonchemotactic strains by insertional mutagenesis. The arginine-requiring strain arg7-8 was transformed with DNA carrying the wild-type ARG7 gene. Of the 8630 arginine-independent transformants obtained, five are defective in their chemotaxis towards various sugars. Two of the mutants (CTX2 and CTX3) are blocked only in their response to xylose. Mutant CTX1 is blocked in its response to xylose, maltose and mannitol, but displays normal taxis to sucrose. Mutants CTX4 and CTX5 lack chemotactic responses to all sugars tested. CTX1, CTX4 and CTX5 represent novel chemotactic phenotypes not previously obtained using ultra-violet or chemical mutagenesis. Genetic analysis confirms that each mutation maps to a single nuclear locus that is unlinked to the mating-type locus. Further analysis of CTX4 indicates that the mutant allele is tagged by the transforming ARG7 DNA. CTX4 appears to be defective in a component specific for chemotactic signal transduction since it exhibits wild-type photobehavioural responses (phototaxis and photoshock) as well as the wild-type responses of EGTA-induced trans-flagellum inactivation and acid-induced deflagellation. Insertional mutagenesis has thus permitted the generation of novel chemotactic mutants that will be of value in the molecular dissection of the signalling machinery.     

Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis

Three out of 10 Helicobacter pylori clinical isolates were found to be naturally competent for genetic transformation to streptomycin resistance by chromosomal DNA extracted from a spontaneous streptomycin-resistant H. pylori mutant. The frequency of transformation varied between 5 × 10^?4 and 4 × 10^?6, depending on the H. pylori isolate used. Transposon shuttle mutagenesis based on this natural competence was established using the flagellin gene flaA as the target. The cloned flaA gene was interrupted by insertion of TnMax1, a mini-Tn1721 transposon carrying a modified chloramphenicol-acetyltransferase gene, the cat_GC cassette. Natural transformation of competent H. pylori strains with plasmid constructs harbouring a cat_GC-inactivated flaA gene resulted in chloramphenicol-resistant transformants at an average frequency of 4 × 10^?5. Southern hybridization experiments confirmed the replacement of the chromosomal H. pylori flaA gene by the cat-inactivated cloned gene copy via homologous recombination resulting in allelic exchange. Phenotypic characterization of the mutants demonstrated the absence of flagella under the electron microscope and the loss of bacterial motility. Immunoblots of cell lysates of the H. pylori mutants with an antiserum raised against the C-terminal portion of recombinant H. pylori major flagellin (FlaA) confirmed the absence of the 54kDa FlaA protein. This efficient transposon shuttle mutagenesis procedure for H. pylori based on natural competence opens up new possibilities for the genetic assessment of putative H. pylori virulence determinants.     

Mutations at the Asc locus of tomato confer resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The fungal pathogen Alternaria alternata f. sp. lycopersici produces host-selective AAL-toxins that cause Alternaria stem canker in tomato. Susceptibility to the disease is based on the relative sensitivity of the host to the AAL-toxins and is controlled by the Asc locus on chromosome 3L. Chemical mutagenesis was employed to study the genetic basis of sensitivity to AAL-toxins and susceptibility to fungal infection. Following the treatment of seeds of a susceptible line with ethyl methanesulphonate (EMS), resistant M₂ mutants were obtained. Most plants with induced resistances showed toxin-sensitivity responses that were comparable to those of resistant control lines carrying the Asc locus. In addition, genetic analysis of the mutagenised plants indicated that the mutations occurred at the Asc locus. Furthermore, novel mutants were identified that were insensitive to the AAL-toxins at the seedling stage but toxin-sensitive and susceptible to fungal infection at mature stages. No AAL-toxin-insensitive insertion mutants were identified following a transposon mutagenesis procedure. Molecular mechanisms involved in host defence against A a. lycopersici are discussed.