Tn4563 transposition in Streptomyces coelicolor and its application to isolation of new morphological mutants.

The Tn3-like transposon Tn4556 (and its derivatives Tn4560 and Tn4563) has been used for insertion mapping of genetic loci cloned on plasmids, but it has been difficult to obtain chromosomal insertions, largely because of the lack of a strong selection against transposon donor molecules. In this communication, we report two efficient selection techniques for transposition and their use in the isolation of chromosomal insertion mutations. A number of independent Streptomyces coelicolor morphological mutants (bld and whi) were obtained. Two of the bld mutations were mapped to locations on the chromosome by SCP1-mediated conjugation; at least one mutation, bld-5m1, appears to define a novel locus involved in control of S. coelicolor morphogenesis and antibiotic production.     

High frequency of independent IS50 transposition during Tn5 mutagenesis of Bordetella pertussis virulence-associated genes

Transposon Tn5-generated mutants of Bordetella pertussis were selected on the basis of their inability to bind the dye Congo red (Crb-). No mutants which were solely Crb- were found. Ten mutants were phenotypically equivalent to previously described strains with mutations in the virulence regulatory (bvg) locus and failed to express a range of virulence-associated factors. Two of these mutants were shown to have Tn5 insertions within the bvg locus, while another two mutants showed deletions in this regulatory region. Complementation studies indicated that the other six mutants had spontaneous mutations in the bvg locus, but with Tn5 inserted elsewhere in the chromosome. Several of the mutants, besides having a single Tn5 insertion, also showed additional IS50 insertions, indicating that the IS50 element contained within Tn5 had transposed independently. Such additional insertion events, which themselves would have the potential to cause mutation, could complicate the interpretation of mutant phenotypes which could thus arise from the insertional inactivation of more than one gene.     

Construction of a physical map of the chromosome of Shigella flexneri 2a and the direct assignment of nine virulence-associated loci identified by Tn5 insertions

To establish the molecular basis of the chromosomal virulence genes of Shigella flexneri 2a (YSH6000), a Notl restriction map of the chromosome was constructed by exploiting Notl-linking clones, partial Notl digestion and DNA probes from various genes of Escherichia coli K-12. The map revealed at least three local differences in the placements of genes between YSH6000 and E. coli K-12. Using the additional Notl sites introduced by Tn5 insertion, nine virulence loci identified previously by random Tn5 insertions were physically mapped on the chromosome. To demonstrate the versatility of the Notl map in direct assignment of the virulence loci tagged by Tn5 to a known genetic region in E. coli K-12, the major class of avirulent mutants defective in the core structure of lipopolysaccharide (LPS) was examined for the sites of Tn5 insertions. The two Notl segments created by the Tn5 insertion in the Notl fragment were analysed by Southern blotting with two DNA probes for the 5' and 3' flanking regions of the rfa region, and shown to hybridize separately with each of them, confirming the sites of Tn5 in the rfa locus. This approach will facilitate direct comparison genetically mapped Tn5 insertion mutations of S. flexneri with genes physically determined in E. coli K-12.     

Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58.

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.     

Virulence-associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis

Shigellae are the causative agents of bacillary dysentery and are capable of invading epithelial cells, multiplying therein and spreading into adjacent cells. To identify genes on the chromosome associated with the virulence phenotype, 9114 independent Tn5 insertion mutants were isolated in a virulent strain of Shigella flexneri. By using an in vitro assay for intercellular spread or an animal infection model, the Serény test, 50 chromosomal Tn5 mutants with reduced virulence were identified. The 50 mutants were characterized with respect to their virulence phenotypes, including three different mutations that affect invasion of epithelial cells, bacterial metabolism and structure of lipopolysaccharide. Mutants with reduced invasive ability were further characterized and it was found that two of them had decreased levels of IpaB, C and D antigens as well as the mRNA for the ipaBCD operon encoded by the large virulence plasmid, suggesting that positive regulatory elements for the ipaBCD operon are encoded by the chromosome. Assignment of the 50 Tn5 insertions of the mutants to the 19 NotI restriction fragments of the chromosomal DNA has permitted the identification of at least nine virulence-associated chromosomal loci.     

Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus

A Rhizobium loti gene required for effective invasion of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis. Cosmids that complemented a previously isolated mutation (239) at this invasion (inv) locus were identified by in planta complementation and used to construct a physical map of the gene region. The insertion site of Tn5 in PN239 was mapped to a 7.5-kb EcoRI fragment, which complemented the mutation when subcloned into pLAFR1. Further Tn5 mutagenesis of the 7.5-kb fragment was carried out in Escherichia coli using bacteriophage lambda 467, and the mutations homogenotized into R. loti NZP2037. Three additional Fix- mutations were isolated, and these were found to map adjacent to the position of the original mutation in strain PN239. All the other Tn5 insertions isolated in the 7.5-kb fragment gave a Fix+ phenotype on L. pedunculatus. Electron microscopic examination of the L. pedunculatus nodules induced by the isolated Fix- mutants showed that bacteria were either blocked in release from the infection threads or were unable to undergo normal bacteroid development. The inv locus as defined by the Tn5 insertions was sequenced, and a single open-reading frame (ORF) of 576 bp, corresponding to a polypeptide of 21.3 kDa, was identified. The position and orientation of this ORF were consistent with those of the isolated Tn5 Fix- insertions.     

Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism.

Mutants of Agrobacterium tumefaciens which affect virulence or the ability to catabolize octopine were isolated after Tn5-induced mutagenesis. Of 8,900 colonies tested, 7 mutants with Tn5 insertions in a specific region of other Ti plasmid unable to catabolize octopine were isolated. Thirty-seven mutants affected in tumorigenesis resulted from insertions in the Ti plasmid and the Agrobacterium chromosome. Of these mutations, 12 were chromosomal and 25 mapped on the plasmid. Twenty-three mapped within a 20-megadalton region, which is distinct from the Ti plasmid sequences found stably integrated into the plant cell genome T-deoxyribonucleic acid). Included in these were mutants that were either a virulent or produced tumors with unusual morphologies. Three mutants contained insertions in the T-deoxyribonucleic acid. These three mutants incited tumors which synthesized octopine but had an altered morphology due to either extensive proliferation of shoots or roots from the tumor callus. Three additional mutants not caused by Tn5 contained mutations in the Ti plasmid.     

Use of transposon-transposase complexes to create stable insertion mutant strains of Francisella tularensis LVS

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 x 10(-8) +/- 0.87 x 10(-8) per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.     

"Frizzy" mutants: a new class of aggregation-defective developmental mutants of Myxococcus xanthus

During fruiting-body formation in Myxococcus xanthus, cells aggregate into raised mounds, where they sporulate. A new class of aggregation-defective developmental mutants was identified within a collection of nonfruiting mutants of M. xanthus. The mutants failed to aggregate into discrete mounds, but rather aggregated into "frizzy" filaments. Many cells within the filaments sporulated normally. Pairwise mixtures of representative frizzy mutants were unable to stimulate each other to aggregate normally. Two strains of M. xanthus were isolated which contained transposon Tn5 insertions mapping near one frizzy mutation. A search through 36 mutants exhibiting the frizzy phenotype showed that all were linked to the same Tn5 insertion sites. Three-factor cross-analysis of 22 of these mutants allowed the mapping of these mutations into many loci. The localization of Tn5 inserts adjacent to this region make possible further manipulation of these genes.     

Spontaneous transposition in the bacteriophage lambda cro gene residing on a plasmid.

A new mutagenesis assay system based on the phage lambda cro repressor gene residing on a plasmid was developed. The assay detects mutations in cro that decrease the binding of the repressor to the OR operator in an OR PR-lacZ fusion present in a lambda prophage. Mutations arose spontaneously during growth of E. coli cells harboring cro plasmids at a frequency of 3-6 x 10(-6). Analysis of some 200 cro mutants from several 'wild-type' strains revealed a substantial fraction of 25-70% insertion events caused by transposition of IS elements. Most of the insertions were caused by IS1, but IS5 insertions were observed too. In strains harboring Tn10, IS10 was responsible for most insertions. Restriction nuclease digestion analysis revealed a preference for insertion of IS10 into the C-terminal half of cro, despite the absence of sequences which are known hot spots for Tn10 insertions. The frequency of IS1 insertions into cro decreased 25-60-fold and that of IS10 insertions decreased 200-fold in cells carrying the recA56 mutation, suggesting that RecA is involved in transposition of these elements. During the logarithmic phase of growth, the mutation frequency was constant for at least 22 generations; however, upon continuous incubation at the stationary phase, the mutation frequency gradually increased, yielding a 3-fold increase in the frequency of insertion and a 4-5-fold increase in point mutation. Genomic Southern analysis of chromosomal IS elements in cells which underwent a transposition from the chromosome into the cro plasmid revealed that the number and distribution of IS1 and IS5 were usually unaltered compared to cells which did not undergo a transposition event. In contrast, essentially each IS10 transposition was accompanied by multiple events which led to changes in the number and distribution of chromosomal IS10 elements.     

The mdoA locus of Escherichia coli consists of an operon under osmotic control

In Escherichia coli, the 5 kb mdoA locus is involved in the osmotically controlled biosynthesis of periplasmic membrane-derived oligosaccharides (MDOs). The structure of this locus was analysed by in vitro cassette insertion, transposon mutagenesis, and gene-fusion analysis. A 'neo' cassette, derived from the neomycin phosphotransferase II region of transposon Tn5, was inserted into mdoA, borne by a multicopy plasmid. This plasmid was shown to complement two previously described mdoA mutations, depending on the orientation of the exogenous gene. Thus, the gene altered by these mutations could be expressed under the control of the exogenous promoter. Moreover, the 'neo' cassette inactivated another, uncharacterized, mdo gene, because when this insertion was transferred into the chromosome MDO synthesis was abolished. The existence of a second gene was confirmed by complementation analysis with a collection of Tn1000 insertions into mdoA. Two groups were defined, and the two genes are organized into an operon (mdoGH). This conclusion was reached because Tn1000 insertions in the first gene displayed a polar effect on the expression of the second gene. An active gene fusion was obtained on a multicopy plasmid between the beginning of mdoH and lacZ. The hybrid beta-galactosidase activity followed the same osmotically controlled response as that described for of MDO synthesis. This regulation was unaffected by the presence, or absence, of MDOs in the periplasm. Finally, the amount of mdoA-specific mRNAs, determined by dot blot hybridization, decreased when the osmolarity of the growth medium increased.     

Ty1 Transposition in Saccharomyces Cerevisiae Is Nonrandom

A large collection of Ty1 insertions in the URA3 and LYS2 loci was generated using a GAL1-Ty1 fusion to augment the transposition frequency. The sites of insertion of most of these Ty elements were sequenced. There appears to be a gradient of frequency of insertion from the 5' end (highest frequency) to the 3' end (lowest frequency) of both loci. In addition we observed hotspots for transposition. Twelve of the 82 Ty1 insertions in the URA3 locus were inserted in exactly the same site. Hotspots were also observed in the LYS2 locus. All hotspots were in the transcribed part of the genes. Alignment of the sites of insertion and of the neighboring sequences only reveals very weak sequence similarities.     

Identification of DNA regions homologous to nitrogen fixation genes nifE, nifUS and fixABC in Azospirillum brasilense Sp7

A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nifgene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.