Effects of antibiotics in soil on the population dynamics of transposon Tn5 carrying Pseudomonas fluorescens

The effects of kanamycin and streptomycin added to soil on the survival of transposon Tn5 modified Pseudomonas fluorescens strain R2f were investigated. Kanamycin in high (180 g g^-1 dry soil) or low (18 g g^-1) concentration or streptomycin in low concentration in Ede loamy sand soil had no noticeable effect on inoculant population dynamics in soil and wheat rhizosphere, whereas streptomycin in high concentration had a consistent significant stimulatory effect, in particular in the wheat rhizosphere. Streptomycin exerted its effect by selecting P. fluorescens with Tn5 insertion whilst suppressing the unmodified sensitive parent strain, as evidenced by comparing the behaviour of these two strains in separate and mixed inoculation studies.Soil textural type influenced the effect of streptomycin on the Tn5 carrying inoculant; the effect was consistently detected in rhizosphere and rhizoplane samples of wheat grown in Ede loamy sand after 7 and 14 days incubation, whereas it was only apparent after 7 days in rhizoplane or rhizosphere (and bulk soil) samples of wheat grown in two silt loam soils. Modification of soil pH by the addition of CaCO₃ or bentonite clay resulted in an enhancement of the selective effect of streptomycin by CaCO₃ and its abolishment by bentonite clay.The addition to soil of malic acid or wheat root exudate, but not of glucose, enhanced the streptomycin selective effect on the Tn5-modified P. fluorescens strain. Neither the streptomycin producer Streptomyces griseus nor two non-inhibiting mutants obtained following UV irradiation affected the dynamics of P. fluorescens (chr::Tn5) in soil and wheat rhizosphere.The effect of streptomycin in soil on inoculant Tn5 carrying bacteria depends on conditions such as soil type, the presence of (wheat) root exudates and the type of available substrate.     

Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli

Summary The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).     

Precise excision of bacterial transposon Tn5 in yeast

Summary We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.All insertions inactivated the LYS2 gene and were able to revert with low (about 10^-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.     

Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons.

Summary Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA ^– background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5 CTAAAACAAAGTTA 3 (hs2). The double-site-specific recombination occurred with a frequency of 10^–5–10^–6. The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a trans-acting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.     

Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene

Summary A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons. This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup. Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution. In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655. Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class 11 transposons, including the Tn1721 subgroup. Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.Abbreviations Ap ampicillin - Cb carbenicillin - Cm chloramphenicol - Km kanamycin - Sm streptomycin - Tc tetracycline - Tp trimethoprim     

Cloning and characterisation of the recA gene of Agrobacterium tumefaciens C58

Summary A gene library of Agrobacterium tumefaciens C58 has been constructed in the plasmid vector pACYC184. A recombinant plasmid was isolated from the library by interspecific complementation in E. coli, which contained the A. tumefaciens recA gene. Heterologous Southern blotting and DNA sequence analysis have demonstrated the existence of considerable homology between the recA genes of A. tumefaciens, E. coli and R. meliloti.Abbreviations MMS methyl methanesulfonate - UV ultraviolet light - bp base pairs - kbp kilo base pairs - dATP deoxyadenosine 5-triphosphate - dNTP deoxynucleoside triphosphate - Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tet tetracycline     

Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21

Summary A new gene for trimethoprim resistance, dhfrV, found in several plasmid isolates with different characteristics, was sequenced and found to correspond to a peptide of 157 amino acids showing 75% similarity with the previously characterized, drug resistant dihydrofolate reductase of type I. The sequenced surroundings of dhfrV in plasmid pLMO20, were found to be almost identical with genetic areas surrounding resistance genes in transposon Tn21 and in R plasmid R388. The trimethoprim resistance genes of pLMO20 and R388 and the spectinomycin resistance gene of Tn21 could be regarded as having been inserted, by recombination, into an evolutionary older structure containg the sulfonamide resistance gene, sulI. The latter gene was sequenced and found to correspond to a peptide of 279 amino acids and with a molecular weight of 30126 daltons. The inserted genes were found to be governed by a promoter situated in the highly conserved structure and also controlling expression of sulI. The insertion points of the different resistance genes were precisely defined, and at the 3 ends of the inserted genes inverted repeats allowing the formation of stem and loop structures were found. Similar structures were found at the 3 ends of the antibiotic resistance genes in Tn7, which could indicate similar recombination mechanisms to be effective in the evolutionary construction of all these different resistance elements.     

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation

Summary Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod^-) or nitrogen fixation (Fix^-). Seven Nod^- mutants were isolated from strain USDA 110 and from strain 61 A101 C, 4 Nod^- mutants and 14 Fix^- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His^- Nod^- mutants of USDA 110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix^- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three nonauxotrophic Nod^- mutants and one His^-Nod^- mutant of USDA 110. Homogenotization of the cloned fragments into wild-type strain USDA 110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod^- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.     

Molecular cloning of protease gene(s) from Xanthomanas campestris pv. campestris: Expression in Escherichia coli and role in pathogenicity

Summary The recombinant plasmid pIJ3070 isolated from a genomic library of Xanthomonas campestris pv. campestris constructed in the conjugal cosmid pLAFR3 contains protease gene(s) which can be expressed in Escherichia coli. Tn5 mutagenesis and subcloning revealed that the protease structural gene(s) is(are) located in a ca. 10 kb EcoRI fragment. Several protease-minus mutants of X. c. campestris were obtained by Tn5 mutagenesis of pIJ3070 and marker exchange techniques. Studies of pathogenicity of these Tn5 mutants showed that the protease is not critically important for the pathogenicity of X. c. campestris on turnip plants but may play a minor role in disease development.Abbreviations Gm gentamicin - Km kanamycin - Rif rifampicin - Spc spectinomycin - Sm streptomycin - Tc tetracycline     

Chloroplast targeting of spectinomycin adenyltransferase provides a cell-autonomous marker for monitoring transposon excision in tomato and tobacco

Antibiotic resistance genes can act as either cell autonomous or non-cell autonomous genetic markers with which to monitor the excision of plant transposons. To convert spectinomycin resistance from a noncell autonomous resistance to cell autonomous resistance, a transit peptide for chloroplast localization from a petunia ribulose bisphosphate carboxylase (rbcS) gene was fused in-frame to the aadA gene, which confers spectinomycin and streptomycin resistance. Constructs were generated in which the expression of this chimeric gene was prevented by the presence, in the 5 untranslated leader, of the maize transposons Activator (Ac) or Dissociation (Ds). When progeny of tobacco or tomato plants transformed with these constructs were germinated on spectinomycin-containing medium, germinally revertant and somatically variegated individuals could be distinguished.     

Construction of a transposon containing a gene for polygalacturonate trans-eliminase from Klebsiella oxytoca

A DNA fragment containing a Klebsiella oxytoca gene for polygalacturonate trans-eliminase was cloned into the kanamycin resistance transposon Tn5. This new transposon, designated Tn5-Pga ⁺, had a transposition frequency of 1×10^-6. The broad host range plasmid pR751::Tn5-Pga ⁺ was conjugally transferred to a variety of genetic backgrounds. The ability to degrade polygalacturonate was expressed in Aeromonas hydrophila, Alcaligenes eutrophus, Azotomonas insolita, Escherichia coli, Pseudomonas putida and Rhodopseudomonas sphaeroides, but not in Zymomonas mobilis.Abbreviations PGA polygalacturonate - UGA unsaturated galacturonic acid - PATE polygalacturonate trans-eliminase - PG polygalacturonase - CVP crystal-violet pectate     

The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector

Summary A plant gene vector cassette to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.Abbreviations OCS octopine synthase (gene) - NOS nopaline synthase (gene) - NPT-II neomycin phosphotransferase (gene) of transposon Tn5 - vir Ti-plasmid region encoding virulence functions - Cb carbenicillin - Gm gentamycin - Km kanamycin - Cm chloramphenicol - Sm streptomycin - Sp spectinomycin - Rif rifampicin - Ery erythromycin - bom basis of mobilization - ori _r origin of conjugational plasmid transfer - Tra, Mob functions required for conjugational transfer of plasmids - BAP N⁶-benzylaminopurine - NAA -naphthaleneacetic acid - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide     

Localization of his genes on the Rhizobium trifolii RS800 linkage map

Summary We have studied N-methyl-N-nitro-N-nitrosoguanidine (NTG)- and Tn5-induced histidine auxotrophic mutants in Rhizobium trifolii. HisGE, hisD and hisH mutants have been characterized. Using the Kemper's equation we have located them on the R. trifolii linkage map. The hisGE and hisD genes are clustered in the same region and are closely linked to the spectinomycin marker. The hisH gene is located in another region equidistant from the streptomycin and rifampicin markers. The two regions carrying his genes are separated by a distance approximately one-third of the length of the chromosome.     

A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM-3.

Summary The genetic environment of plasmid-borne bla _TEM mutant genes, encoding nine distinct TEM-type extended-spectrum -lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine bla _TEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes bla _TEM-1 or _–2. A 6.6 kb DNA fragment of pCFF04 containing bla _TEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that bla _TEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was, in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with bla _TEM-3.     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.     

Nisin biosynthesis genes are encoded by a novel conjugative transposon

Summary Genes for biosynthesis of the lactococcal peptide antibiotic nisin were shown to be encoded by a novel chromosomally located transposon Tn5301. The element is 70 kb in size and lacks inverted repeats at its termini. Although a copy of the insertion sequence IS904 is located near to one end, this did not appear to be involved in the transposition process. The integrated element is flanked by the directly repeated sequence 5-TTTTTG-3. Analysis of ten independent transconjugants revealed that Tn5301 integration is site-specific; two chromosomal targets were identified and shown to have some sequence homology. The element shares features with the Tn916 family of conjugative transposons and with Tn554 but is also exhibits some unique properties. Tn5301 is thus considered to be the prototype of a novel class of conjugative transposon.     

Genetic analysis of a transposon carrying toluene degrading genes on a TOL plasmid pWWO

Summary Toluene degrading (xyl) genes on a Pseudomonas TOL plasmid pWWO are located within a 39-kb DNA portion. The 56-kb region including these xyl genes and its 17-kb derivative with a deletion of the internal 39-kb portion transposed to various sites on target replicons such as pACYC184 and R388 in escherichia coli recA strains. Thus the 56- and 17-kb regions were designated Tn4651 and Tn4652, respectively. Genetic analysis of Tn4652 demonstrated that its transposition occurs by a two-step process, namely, cointegrate formation and its subsequent resolution. The presence in cis of DNA sequences of no more than 150 bp at both ends of Tn4652 was prerequisite for cointegrate formation, and this step was mediated by a trans-acting factor, transposase, which was encoded in a 3.0-kb segment at one end of the transposon. Cointegrate resolution took place site-specifically within a 200-bp fragment, which was situated 10 kb away from the transposase gene. Based on the stability of cointegrates formed by various mini-Tn4652 derivatives, it was shown that the cointergrate resolution requires two trans-acting factors encoded within 1.0- and 1.2-kb fragments that encompass the recombination site involved in the resolution.     

Transposition and transduction of plasmid DNA inStreptomyces spp.

Summary To expand the application of molecular genetics to many different streptomycete species, we have been developing two potentially widely applicable methodologies: transposon mutagenesis and plasmid transduction. We constructed three transposons from theStreptomyces lividans insertion sequence IS493. Tn5096 and Tn5097 contain an apramycin resistance gene inserted in different orientations between the two open reading frames of IS493. These transposons transpose from different plasmids into many different sites in theStreptomyces griseofuscus chromosome and into its resident linear plasmids. Tn5099 contains a promoterlessxylE gene and a hygromycin-resistance gene inserted in IS493 close to one end. Tn5099 transposes inS. griseofuscus giving operon fusions in some cases that drive expression of thexylE gene product, catechol deoxygenase, giving yellow colonies in the presence of catechol. We have also developed plasmid vectors that can be transduced into many streptomycete species by bacteriophage FP43. We describe the characterization of FP43 and mapping of several bacteriophage functions. The region of cloned FP43 DNA essential for plasmid transduction includes the origin for headful packaging.     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.